Light-activated chimeric opsins and methods of using the same

ABSTRACT

Provided herein are compositions comprising light-activated chimeric proteins expressed on plasma membranes and methods of using the same to selectively depolarize excitatory or inhibitory neurons.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/US2011/059276, filed Nov. 4, 2011, and claims priority to U.S.Provisional Patent Application Nos. 61/410,736 filed on Nov. 5, 2010;61/410,744 filed on Nov. 5, 2010; and 61/511,912 filed on Jul. 26, 2011,the disclosures of each of which are incorporated herein by reference intheir entireties.

TECHNICAL FIELD

This application pertains to compositions comprising animal cellsexpressing light-activated chimeric proteins on their plasma membranesand methods of using the same to selectively depolarize excitatory orinhibitory neurons residing in the same microcircuit in the pre-frontalcortex.

BACKGROUND

The neurophysiological substrates of most psychiatric disorders arepoorly understood, despite rapidly emerging information on geneticfactors that are associated with complex behavioral phenotypes such asthose observed in autism and schizophrenia (Cichon et al., The AmericanJournal of Psychiatry 166(5):540 (2009); O'Donovan et al., HumanGenetics 126(1): 3 (2009)). One remarkable emerging principle is that avery broad range of seemingly unrelated genetic abnormalities can giverise to the same class of psychiatric phenotype (such as social behaviordysfunction; Folstein & Rosen-Sheidley, Nature Reviews 2(12):943(2001)). This surprising pattern has pointed to the need to identifysimplifying circuit-level insights that could unify diverse geneticfactors under a common pathophysiological principle.

One such circuit-level hypothesis is that elevation in the ratio ofcortical cellular excitation and inhibition (cellular E/I balance) couldgive rise to the social and cognitive deficits of autism (Rubenstein,Current Opinion in Neurology 23 (2):118; Rubenstein & Merzenich, Genes,Brain, and Behavior 2(5):255 (2003)). This hypothesis could potentiallyunify diverse streams of pathophysiological evidence, including theobservation that many autism-related genes are linked togain-of-function phenotypes in ion channels and synaptic proteins(Bourgeron, Current Opinion in Neurobiology 19 (2), 231 (2009)) and that˜30% of autistic patients also show clinically apparent seizures(Gillberg & Billstedt, Acta Psychiatrica Scandinavica, 102(5):321(2000)). However, it has not been clear if such an imbalance (to berelevant to disease symptoms) would be operative on the chronic (e.g.during development) or the acute timescale. Furthermore, this hypothesisis by no means universally accepted, in part because it has not yet beensusceptible to direct testing. Pharmacological and electricalinterventions lack the necessary specificity to selectively favoractivity (in a manner fundamentally distinct from receptor modulation)of neocortical excitatory cells over inhibitory cells, whether in theclinical setting or in freely behaving experimental mammals duringsocial and cognitive tasks. It is perhaps related to challenges such asthis that the social and cognitive deficits of autism and schizophreniahave proven largely unresponsive to conventional psychopharmacologytreatments in the clinic.

Optogenetics is the combination of genetic and optical methods used tocontrol specific events in targeted cells of living tissue, even withinfreely moving mammals and other animals, with the temporal precision(millisecond-timescale) needed to keep pace with functioning intactbiological systems. The hallmark of optogenetics is the introduction offast light-activated channel proteins to the plasma membranes of targetneuronal cells that allow temporally precise manipulation of neuronalmembrane potential while maintaining cell-type resolution through theuse of specific targeting mechanisms. Among the microbial opsins whichcan be used to investigate the function of neural systems are thechannelrhodopsins (ChR2, ChR1, VChR1, and SFOs) used to promotedepolarization in response to light. In just a few short years, thefield of optogenetics has furthered the fundamental scientificunderstanding of how specific cell types contribute to the function ofbiological tissues such as neural circuits in vivo. Moreover, on theclinical side, optogenetics-driven research has led to insights intoParkinson's disease and other neurological and psychiatric disorders.

However, there are limitations to existing optogenetic tools forexploring the hypothesis that elevation in the ratio of cortical E/Ibalance might be associated with the social and cognitive deficits ofautism and other disorders such as schizophrenia. Conventionalchannelrhodopsin photocurrents display significant desensitization whichprecludes the generation of step-like changes in E/I balance (insteadrequiring ramping or pulsing, which would not be suitable forinvestigation of stable changes in cellular E/I balance); moreover, bothSFOs and conventional ChRs are driven by blue light, which precludeswithin-preparation comparison of the effects of driving differentpopulations of circuit elements (such as excitatory and inhibitoryneurons). Therefore, what is needed is a tool that would allow themanipulation of cortical E/I balances and the monitoring of gammaoscillations in cortical slices to permit the investigation of how thesemanipulations affect downstream neurons residing in the samemicrocircuit in the pre-frontal cortex.

BRIEF SUMMARY OF THE INVENTION

Provided herein are compositions comprising chimeric light-activatedprotein cation channels which are capable of mediating a depolarizingcurrent in the cell when the cell is illuminated with light.

Provided herein are animal cells comprising a light-activated proteinexpressed on the cell membrane, wherein the protein is (a) a chimericprotein derived from VChR1 from Volvox carteri and ChR1 fromChlamydomonas reinhardti, wherein the protein comprises the amino acidsequence of VChR1 having at least the first and second transmembranehelices replaced by the first and second transmembrane helices of ChR1;(b) is responsive to light; and (c) is capable of mediating adepolarizing current in the cell when the cell is illuminated withlight. In some embodiments, the cells are isolated or in a cell culturemedium.

Also provided herein is a population of cells comprising the cellexpressing the chimeric protein described herein on the cell membrane.Also provided herein are non-human animals and brain tissue slicescomprising a cell expressing the chimeric protein described herein onthe cell membrane.

Provided herein are polynucleotide comprising a nucleotide sequenceencoding a light activated protein expressed on the cell membrane,wherein the protein is a chimeric protein derived from VChR1 from Volvoxcarteri and ChR1 from Chlamydomonas reinhardti, wherein the proteincomprises the amino acid sequence of VChR1 having at least the first andsecond transmembrane helices replaced by the first and secondtransmembrane helices of ChR1; is responsive to light; and is capable ofmediating a depolarizing current in the cell when the cell isilluminated with light. Vectors (such as expressing vectors) comprisingthe polynucleotides are also provided. In some embodiments, theexpression vector is a viral vector (e.g., an AAV vector, a retroviralvector, an adenoviral vector, a HSV vector, or a lentiviral vector).

Also provided herein are methods of using the animal cells expressingthe chimeric protein described herein on the cell membrane, the methodscomprise activating the chimeric protein with light.

Also provided herein are methods of selectively depolarizing excitatoryor inhibitory neurons residing in the same microcircuit, the methodscomprising: selectively depolarizing an excitatory neuron comprising afirst light-activated protein, wherein the first light activated proteinis depolarized when exposed to light having a first wavelength; orselectively depolarizing an inhibitory neuron comprising a secondlight-activated protein, wherein the second light activated protein isdepolarized when exposed to light having a second wavelength. In someembodiments, the first or the second light activated protein is achimeric protein derived from VChR1 from Volvox carteri and ChR1 fromChlamydomonas reinhardti, wherein the protein comprises the amino acidsequence of VChR1 having at least the first and second transmembranehelices replaced by the first and second transmembrane helices of ChR1.In some embodiments, wherein the first light-activated protein comprisesan amino acid sequence at least 95% identical to the sequence shown inSEQ ID NO: 1, 3, 5, or 7, and wherein the second light-activated proteincomprises an amino acid sequence at least 95% identical to the sequenceshown in SEQ ID NO:11, 12, 13, or 14.

A method of selectively depolarizing excitatory or inhibitory neuronsresiding in the same microcircuit, the method comprising: expressing afirst light-activated protein in an excitatory neuron; and expressing asecond light activated protein in an inhibitory neuron, wherein thefirst light activated protein is independently depolarized when exposedto light having a first wavelength and wherein the second lightactivated protein is independently depolarized when exposed to lighthaving a second wavelength. In some embodiments, the first or the secondlight activated protein is a chimeric protein derived from VChR1 fromVolvox carteri and ChR1 from Chlamydomonas reinhardti, wherein theprotein comprises the amino acid sequence of VChR1 having at least thefirst and second transmembrane helices replaced by the first and secondtransmembrane helices of ChR1. In some embodiments, the firstlight-activated protein comprises an amino acid sequence at least 95%identical to the sequence shown in SEQ ID NO: 1, 3, 5, or 7, and whereinthe second light-activated protein comprises an amino acid sequence atleast 95% identical to the sequence shown in SEQ ID NO:11, 12, 13, or14.

Also provided herein are methods for identifying a chemical compoundthat selectively inhibits the depolarization of excitatory or inhibitoryneurons residing in the same microcircuit, the method comprising: (a)selectively depolarizing an excitatory neuron comprising a firstlight-activated protein with light having a first wavelength orselectively depolarizing an inhibitory neuron comprising a secondlight-activated protein with light having a second wavelength; (b)measuring an excitatory post synaptic potential (EPSP) in response toselectively depolarizing the excitatory neuron comprising a firstlight-activated protein or measuring an inhibitory post synaptic current(IPSC) in response to selectively depolarizing an inhibitory neuroncomprising a second light-activated protein; (c) contacting theexcitatory neuron or the inhibitory neuron with a chemical compound; (d)measuring the excitatory post synaptic potential (EPSP) or measuring theinhibitory post synaptic current (IPSC) to determine if contactingeither the excitatory neuron or the inhibitory neuron with the chemicalcompound selectively inhibits the depolarization of either neuron. Insome embodiments, the first or the second light activated protein is achimeric protein derived from VChR1 from Volvox carteri and ChR1 fromChlamydomonas reinhardti, wherein the protein comprises the amino acidsequence of VChR1 having at least the first and second transmembranehelices replaced by the first and second transmembrane helices of ChR1.In some embodiments, the first light-activated protein comprises anamino acid sequence at least 95% identical to the sequence shown in SEQID NO: 1, 3, 5, or 7, and wherein the second light-activated proteincomprises an amino acid sequence at least 95% identical to the sequenceshown in SEQ ID NO:11, 12, 13, or 14.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-8 depict engineering of an improved red-shifted channelrhodopsinfor combinatorial optogenetics.

FIG. 1 depicts confocal images of cultured hippocampal neuronstransfected with VChR 1-eYFP or C1V1-eYFP under the control of theCaMKIIa promoter. Box denotes region expanded in the last panel, showingdendritic membrane localization of C1V1-tsYFP. Scale bars: 20 μm (left),4 μm (right).

FIG. 2 depicts peak photocurrents from whole-cell patch clamp recordingsin-cultured hippocampal neurons expressing indicated opsins.

FIG. 3 depicts sequence alignment of ChR1 (SEQ ID NO:16), ChR2 (SEQ IDNO:11) and VChR1 (SEQ ID NO:17). Splice sites for two C1V1 variants areindicated. Putative transmembrane helices 1-7 are indicated with bars(TM1-7); mutated amino acids indicated in grey.

FIG. 4 depicts photocurrent amplitudes recorded in HEK cells expressingC1V1 splice variants 1 and 2.

FIG. 5 depicts single confocal plane images of cultured hippocampalneurons transfected with indicated opsins, fused to EYFP. DNAconcentration was matched across constructs.

FIG. 6 depicts action spectra of ChR2, VChR1, C1V1 wt, C1V (E122T), C1V1(E162T), and C1V1 (E122T/E162T). Photocurrents were collected with 2 mslight pulses in HEK293 cells.

FIG. 7 depicts ion permeance of C1V1 splice variant 1 as measured byphotocurrent magnitude at −40 mV in HEK cells by whole cell patch clampusing cation-isolating external solutions. Data were normalized to themaximum peak Na current.

FIG. 8 depicts a schematic of the C1V1 chimera with point mutationpositions indicated in white. ChR1 sequence indicated with black; VChR1sequence with grey.

FIG. 9-12 depict testing an improved red-shifted channelrhodopsin forcombinatorial optogenetics.

FIG. 9 depicts representative traces and summary plot of channel closuretime constant (τ_(off)) in cultured neurons expressing the indicatedopsins; traces are normalized to peak current.

FIG. 10 depicts C1V1-E122T inactivation compared to deactivation ofChR2.

FIG. 11 depicts inactivation of current in C1V1 double mutantE122T/E162T versus other C1V1 variants.

FIG. 12 depicts mean peak photocurrents recorded in cultured neuronsexpressing the indicated opsins in response to a 2 ms 542 nm lightpulse.

FIG. 13-20 depict photocurrents from acute slice recordings inprefrontal pyramidal neurons.

FIG. 13 depicts that peak photocurrents show consistent correlation withintegrated fluorescence intensity.

FIG. 14 depicts fluorescence-photocurrent relationship in ChR2(H134R)and C1V1(E122T/E162T). Black lines are linear fits to the data.

FIG. 15 depicts acute slice recordings in prefrontal pyramidal neuronsstimulated with 560 nm light pulse trains or current injections at theindicated frequencies. Summary graph shows population data (n=6).

FIG. 16 depicts fraction of successful spikes to current injections (200pA, 10 ms pulses; top left) or 2 ms light pulses at the indicatedwavelengths and light power densities. All pulse trains consisted of20×2 ms pulses delivered through the microscope objective using a SutterDG-4 light source, filtered using 20 nm bandpass filters and additionalneutral density filters to attenuate light power (n=6 cells in 2slices).

FIG. 17 depicts voltage-clamp responses to 542 nm and 630 nm lightpulses in cells expressing C1V1-E122T or C1V1-E 122T/E 162T (top).Current-clamp recording in a C1V1-E 122T expressing cell shows spikingin response to a 5 Hz train of 50 ms 630 nm light at 3.2 mW mm⁻²(bottom).

FIG. 18 depicts kinetics of red light response in C1V1(E122T).Activation time constants (τ_(on)) of photocurrents recorded fromcultured neurons expressing C1V1(E122T) at 540 nm and 630 nm. Note thatlight powers were 3.2 mW mm-2 at 630 nm and 7.7 mW mm⁻² at 540 nm (n=5cells, p=0.0006 paired t-test).

FIG. 19 depicts that voltage clamp traces show responses in a neuronexpressing C1V1(E122T) to 630 nm light pulses. Pulse lengths areindicated above traces. τ_(on) calculated from the 150 ms trace is 67ms.

FIG. 20 depicts current clamp recording from a neuron expressingC1V1(E122T) showing spikes elicited by 50 ms pulses at 630 nm (powerdensity 3.2 mW mm⁻²)

FIG. 21-27 depict independent activation of excitatory pyramidal neuronsand inhibitory parvalbumin-expressing cells.

FIG. 21 depicts current clamp recordings from cultured hippocampalneurons expressing C1V1(E1 22T/E162T) or ChR2(H134R) in response to 2 mslight pulses at 560 nm or 405 nm (5 Hz; 7.6 mW/mm² at both wavelengths).

FIG. 22 depicts a recording configuration in double-injected animalsexpressing C1V1 in cortical pyramidal neurons and ChR2 (H134R) ininhibitory parvalbumin-positive interneurons. To independently expressopsins, PV::Cre mice were injected with a two-virus mix containingLenti-CaMKIIα-C1V1(E122T/E162T) and AAV5-EF1a-DIO-Ch R2 (H 134R).

FIG. 23 depicts voltage clamp recordings from a non-expressing PYRneuron receiving synaptic input from C1V1-expressing PYR-cells andChR2-expressing PV-cells. Clamped at OmV, 405 nm light pulses triggershort-latency IPSCs while 560 nm pulses evoke only small, long-latencyinhibitory synaptic responses.

FIG. 24 depicts voltage clamp recording from the same cell shown in FIG.23. Clamped at −65 mV, 560 nm light pulses trigger EPSCs but 405 nmpulses do not evoke detectable synaptic currents. Gray lines showindividual events; black lines show light pulse-triggered averages.

FIG. 25 depicts an mPFC optrode recording in an anesthetized PV::Cremouse injected with CaMKIIa::C1V1(E162T)-is-eYFP and Ef1a-DIO::ChR2-eYFP(diagram illustrates experimental setup). Violet (405 nm) light pulsesare presented with variable delay (At) relative to green light pulses(example traces).

FIG. 26 depicts a summary graph shows probability of green light-evokedspikes with violet pulses preceding the green light pulses by theindicated delays. Individual points are from single recordings. Blackline shows average for all recordings (>3 recording sites per bin).

FIG. 27 depicts an optrode recording from a mouse injected with virusesshowing one presumed pyramidal unit and one presumed PV unit, firing inresponse to 561 nm stimulation (right, upper waveform) and 405 nmstimulation (right, lower waveform), respectively.

FIG. 28-32 depict combinatorial optogenetic excitation in distinctintact-system preparations.

FIG. 28 depicts combinatorial projection control with C1V1-E122T/E 162Tand ChR2-H134R in vitro. Experimental paradigm showing expression ofC1V1 and ChR2 in cortico-thalamic (CT) and ventrobasal (VB)thalamo-cortical cells (TC), respectively.

FIG. 29 depicts that individual subthreshold inputs from TC and CTfibers lead to spiking in an nRT neuron only when inputs are preciselyco-incident. Delays between CT and TC inputs are indicated on the left.Horizontal dashed lines indicate truncated spikes.

FIG. 30 depicts Voltage-clamp recording from an nRT cell receivingprojections both from CT and TC cells. Simultaneous stimulation (Δt=Oms) leads to a linear summation of evoked EPSCs from both projections.

FIG. 31 depicts normalized number of action potentials (from 6 nRTcells) evoked by CT and TC fibers activated with variable latencies (Δt)indicates that CT and TC inputs lead to effective integration only ifcoincident within 5 ms. Summary data represent mean±SEM.

FIG. 32 depicts temporal precision of C1V1 and ChR2 activation.

FIG. 33-36 depict spectrotemporal separation and combinatorial control:circuit modulation and emergent patterns in altered E/I states underongoing synaptic activity.

FIG. 33 depicts experimental paradigm for SSFO activation of PV neuronsand C1V1 activation in pyramidal neurons.

FIG. 34 depicts voltage clamp recording at 0 mV from a pyramidal neuronin an acute slice preparation from a PV::Cre mouse expressingCaMKIIa::C1V1(E162T) and DIO-SSFO. SSFO and C1V1 are activated by a bluelight pulse (2) and IPSC frequency is increased by sustained SSFOactivity (3; compare upper and lower traces in inset for pre- andpost-activation IPSC activity). A sustained yellow light pulsedeactivates SSFO and activates C1V1 and transiently increases IPSCfrequency (4). Population power spectra (right) show gamma frequencyactivity during optical excitatory neuron activation (590 nm pulse) thatis increased during coactivation of excitatory and PV neurons (470 nmpulse). Diagrams below traces show predicted activity of C1V1 and SSFOduring the experiment.

FIG. 35 depicts that the observed gamma frequency peak was not dependenton prior PV neuron stimulation via SSFO.

FIG. 36 depicts summary IPSC frequencies from FIG. 34 and FIG. 35 atbaseline and after the initial blue or orange pulse. Diagrams belowtraces show predicted activity of C1V1 and SSFO during the experiment.

DETAILED DESCRIPTION

This invention provides, inter alia, compositions comprising animalcells expressing light-activated chimeric proteins on their plasmamembranes and methods of using the same to selectively depolarizeexcitatory or inhibitory neurons residing in the same microcircuit inthe pre-frontal cortex. The inventors have developed chimeric proteinspossessing unique physiochemical properties which for the first timepermit experimental manipulation of cortical E/I elevations and theability to monitor gamma oscillations in cortical slices. These uniquelight-sensitive chimeric proteins can be expressed in either excitatoryor inhibitory neural circuits in the prefrontal cortex of nonhumananimals which can then be depolarized in response to light havingparticular wavelengths. Furthermore, brain slices from non-human animalscontaining cortical excitatory or inhibitory neurons expressing thechimeric light-sensitive proteins disclosed herein can be used to searchfor chemical compounds which can selectively inhibit the depolarizationof either excitatory or inhibitory neurons residing within the sameneural circuit.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,cell biology, biochemistry, nucleic acid chemistry, and immunology,which are well known to those skilled in the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) and MolecularCloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001),(jointly referred to herein as “Sambrook”); Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987, includingsupplements through 2001); PCR: The Polymerase Chain Reaction, (Mulliset al., eds., 1994); Harlow and Lane (1988) Antibodies, A LaboratoryManual, Cold Spring Harbor Publications, New York; Harlow and Lane(1999) Using Antibodies: A Laboratory Manual Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (jointly referred to hereinas “Harlow and Lane”), Beaucage et al. eds., Current Protocols inNucleic Acid Chemistry John Wiley & Sons, Inc., New York, 2000),Handbook of Experimental Immunology, 4th edition (D. M. Weir & C. C.Blackwell, eds., Blackwell Science Inc., 1987); and Gene TransferVectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds., 1987).

DEFINITIONS

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise.

An “animal” can be a vertebrate, such as any common laboratory modelorganism, or a mammal Mammals include, but are not limited to, humans,farm animals, sport animals, pets, primates, mice, rats, and otherrodents.

An “amino acid substitution” or “mutation” as used herein means that atleast one amino acid component of a defined amino acid sequence isaltered or substituted with another amino acid leading to the proteinencoded by that amino acid sequence having altered activity orexpression levels within a cell.

A “chimeric protein” is a protein comprising one or more portionsderived from one or more different proteins. Chimeric proteins may beproduced by culturing a recombinant cell transfected with a nucleic acidthat encodes the chimeric protein.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

V1C1 Chimeric Proteins and Cells Expressing the Same

In some aspects, the animal cells disclosed herein comprise a chimericlight-sensitive protein, known as “C1V1,” which is derived from theVChR1 cation channel from Volvox carteri and the ChR1 cation channelfrom Chlamydomonas Reinhardti. The protein may be comprised of the aminoacid sequence of VChR1, but additionally can have at least the first andsecond transmembrane helices of the VChR1 polypeptide replaced by thecorresponding first and second transmembrane helices of ChR1. C1V1chimeric opsin proteins are assembled from pieces of other opsinproteins that do not express well alone in neurons and which are potent,redshifted, and stable channelrhodopsins. In some embodiments, theanimal cell may express a second light-activated protein on the plasmamembrane of the cell. The second light-activated protein can be capableof mediating a hyperpolarization of the cell plasma membrane in responseto activation by light. Examples of light-activated proteins capable ofmediating a hyperpolarization of the cell plasma membrane can be found,for example, in International Patent Application No: PCT/US2011/028893,the disclosure of which is incorporated by reference herein in itsentirety.

Embodiments of the present disclosure may also be directed towardmodified or mutated versions of C1V1. These proteins can be used aloneor in combination with a variety of other opsins to assert opticalcontrol over neurons. In particular, the use of modified C1V1, inconnection with other opsins, is believed to be useful for opticalcontrol over nervous system disorders. Specific uses of C1V1 relate tooptogenetic systems or methods that correlate temporal, spatial and/orcell type-specific control over a neural circuit with measurablemetrics.

V1C1 Chimeric Proteins

Provided herein are light-activated chimeric proteins expressed on ananimal cell plasma membrane. In some aspects the light-activated proteinis a chimeric protein derived from VChR1 from Volvox carteri and ChR1from Chlamydomonas reinhardti. In some embodiments, the chimeric proteincomprises the amino acid sequence of VChR1 having at least the first andsecond transmembrane helices replaced by the corresponding first andsecond transmembrane helices of ChR1. In other embodiments, the chimericprotein comprises the amino acid sequence of VChR1 having the first andsecond transmembrane helices replaced by the corresponding first andsecond transmembrane helices of ChR1 and further comprises at least aportion of the intracellular loop domain located between the second andthird transmembrane helices replaced by the corresponding portion fromChR1. In some embodiments, the entire intracellular loop domain betweenthe second and third transmembrane helices of the chimericlight-activated protein can be replaced with the correspondingintracellular loop domain from ChR1. In other embodiments, the portionof the intercellular loop domain located between the second and thirdtransmembrane helices that is replaced with the corresponding portion ofChR1 can extend to A145 of SEQ ID NO:1. In other embodiments, thechimeric protein comprises the amino acid sequence of VChR1 having thefirst and second transmembrane helices and the intracellular loop domainreplaced by the corresponding first and second transmembrane helices andintracellular loop domain of ChR1 and further comprises at least aportion of the third transmembrane helix replaced by the correspondingportion of ChR1. In another embodiment, the portion of the thirdtransmembrane helix replaced by the corresponding portion from ChR1 canextend to W163 of SEQ ID NO:1. In some embodiments, the light-activatedchimeric protein comprises the amino acids 1-145 of ChR1 and amino acids102-316 of VChR1. In some embodiments, the light-activated chimericprotein comprises the amino acids 1-162 of ChR1 and amino acids 119-316of VChR1. In some embodiments, the light-activated chimeric protein cancomprise an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ IDNO: 1 without the signal peptide sequence. In some embodiments, thelight-activated chimeric protein can comprise an amino acid sequence atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO: 1

In other embodiments, the light activated chimeric protein is capable ofmediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments the light can be greenlight. In other embodiments, the light can have a wavelength of betweenabout 540 nm to about 560 nm. In some embodiments, the light can have awavelength of about 542 nm. In some embodiments, the chimeric proteinmay not be capable of mediating a depolarizing current in the cell whenthe cell is illuminated with violet light. In some embodiments, thechimeric protein may not be capable of mediating a depolarizing currentin the cell when the cell is illuminated with light having a wavelengthof 405 nm.

In some embodiments, the protein can further comprise a C-terminalfluorescent protein. In some specific embodiments, the C-terminalfluorescent protein can be enhanced yellow fluorescent protein (EYFP),green fluorescent protein (GFP), cyan fluorescent protein (CFP), or redfluorescent protein (RFP). In some embodiments, the light-activatedchimeric protein is modified by the addition of a trafficking signal(ts) which enhances transport of the protein to the cell plasmamembrane. In some embodiments, the trafficking signal is derived fromthe amino acid sequence of the human inward rectifier potassium channelKir2.1. In some embodiments, the trafficking signal comprises the aminoacid sequence KSRITSEGEYIPLDQIDINV. In some embodiments, the signalpeptide sequence in the protein may be replaced with a different signalpeptide sequence.

In some embodiments, the animal cell can be a neuronal cell, a musclecell, or a stem cell. In one embodiment, the animal cell is a neuronalcell. In some embodiments the neuronal cell can be an excitatory neuronlocated in the pre-frontal cortex of a non-human animal. In otherembodiments, the excitatory neuron can be a pyramidal neuron. In stillother embodiments, the inhibitory neuron can be a parvalbumin neuron. Insome embodiments the neuronal cell can be an inhibitory neuron locatedin the pre-frontal cortex of a non-human animal. In some embodiments theneuronal cell can be an inhibitory neuron located in the pre-frontalcortex of a non-human animal.

In some embodiments, the animal cells can further comprise a secondlight-activated protein expressed on the cells' plasma membrane. In someembodiments, the second light-activated protein can be capable ofmediating a hyperpolarizing current in the cell when the cell isilluminated with light. In some embodiments the second light-activatedprotein can be NpHr, eNpHr2.0, eNpHr3.0, eNpHr3.1 or GtR3. Additionalinformation regarding other light-activated cation channels, anionpumps, and proton pumps can be found in U.S. Patent ApplicationPublication Nos: 2009/0093403; and International Patent Application No:PCT/US2011/028893, the disclosures of which are hereby incorporated byreference herein in their entirety. In some embodiments, thelight-activated chimeric protein can have enhanced photocurrents inneural cells exposed to light relative to cells expressing otherlight-activated cation channel proteins. In some embodiments, theenhancement in photocurrent provided by the light-activated chimericprotein can be any of 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, or 15fold, greater than cells expressing other light-activated cation channelproteins, inclusive.

Also provided herein is one or more light-activated proteins expressedon an animal cell plasma membrane, wherein said one or more lightactivated proteins comprises a core amino acid sequence at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 12, 13, or 14 andfurther comprising a trafficking signal (e.g., which enhances transportto the plasma membrane). The trafficking signal may be fused to theC-terminus of the core amino acid sequence or may be fused to theN-terminus of the core amino acid sequence. In some embodiments, thetrafficking signal can be linked to the core amino acid sequence by alinker. The linker can comprise any of 5, 10, 20, 30, 40, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids inlength. The linker may further comprise a fluorescent protein, forexample, but not limited to, an enhanced yellow fluorescent protein, ared fluorescent protein, a green fluorescent protein, or a cyanfluorescent protein. In some embodiments, the trafficking signal can bederived from the amino acid sequence of the human inward rectifierpotassium channel Kir2.1. In some embodiments, the trafficking signalcan comprise the amino acid sequence KSRITSEGEYIPLDQIDINV.

V1C1 Chimeric Mutant Variants

In some aspects, the invention includes polypeptides comprisingsubstituted or mutated amino acid sequences, wherein the mutantpolypeptide retains the characteristic light-activatable nature of theprecursor C1V1 chimeric polypeptide but may also possess alteredproperties in some specific aspects. For example the mutantlight-activated chimeric proteins described herein may exhibit anincreased level of expression both within an animal cell or on theanimal cell plasma membrane; an altered responsiveness when exposed todifferent wavelengths of light, particularly red light; and/or acombination of traits whereby the chimeric C1V1 polypeptide possess theproperties of low desensitization, fast deactivation, low violet-lightactivation for minimal cross-activation with other light-activatedcation channels, and/or strong expression in animal cells.

Light-activated chimeric proteins comprising amino acid substitutions ormutations include those in which one or more amino acid residues haveundergone an amino acid substitution while retaining the ability torespond to light and the ability to control the polarization state of aplasma membrane. For example, light-activated proteins comprising aminoacid substitutions or mutations can be made by substituting one or moreamino acids into the amino acid sequence corresponding to SEQ ID NO:1.In some embodiments, the invention includes proteins comprising alteredamino acid sequences in comparison with the amino acid sequence in SEQID NO:1, wherein the altered light-activated chimeric protein retainsthe characteristic light-activated nature and/or the ability to regulateion flow across plasma membranes of the protein with the amino acidsequence represented in SEQ ID NO:1 but may have altered properties insome specific aspects.

Amino acid substitutions in a native protein sequence may beconservative or non-conservative and such substituted amino acidresidues may or may not be one encoded by the genetic code. The standardtwenty amino acid “alphabet” is divided into chemical families based onchemical properties of their side chains. These families include aminoacids with basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) and sidechains having aromatic groups (e.g., tyrosine, phenylalanine,tryptophan, histidine). A “conservative amino acid substitution” is onein which the amino acid residue is replaced with an amino acid residuehaving a chemically similar side chain (i.e., replacing an amino acidpossessing a basic side chain with another amino acid with a basic sidechain). A “non-conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having achemically different side chain (i.e., replacing an amino acid having abasic side chain with an amino acid having an aromatic side chain). Theamino acid substitutions may be conservative or non-conservative.Additionally, the amino acid substitutions may be located in the C1V1retinal binding pocket, in one or more of the C1V1 intracellular loopdomains, and/or in both the retinal binding pocket or the intracellularloop domains.

Accordingly, provided herein are C1V1 chimeric light-activated proteinsthat may have specific amino acid substitutions at key positionsthroughout the retinal binding pocket of the VChR1 portion of thechimeric polypeptide. In some embodiments, the C1V1 protein can have amutation at amino acid residue E122 of SEQ ID NO:1. In some embodiments,the C1V1 protein can have a mutation at amino acid residue E162 of SEQID NO:1. In other embodiments, the C1V1 protein can have a mutation atboth amino acid residues E162 and E122 of SEQ ID NO:1. In someembodiments, each of the disclosed mutant C1V1 chimeric proteins canhave specific properties and characteristics for use in depolarizing themembrane of an animal cell in response to light.

C1V1-E122 Mutant Polypeptides

Provided herein are the light-activated C1V1 chimeric proteins disclosedherein expressed on an animal cell plasma membrane, wherein one or moreamino acid residues have undergone an amino acid substitution whileretaining C1V1 activity (i.e., the ability to catalyze thedepolarization of an animal cell in response to light activation), andwherein the mutation can be at a glutamic acid residue corresponding toE122 of SEQ ID NO:1 (C1V1-E122). In some embodiments, the C1V1-E122mutant chimeric light-activated protein comprises substitutionsintroduced into the amino acid sequence shown in SEQ ID NO:1 at aminoacid E122 that can result in the chimeric protein having increasedsensitivity to light, increased sensitivity to particular wavelengths oflight, and/or increased ability to regulate the polarization state ofthe plasma membrane of the cell relative to C1V1 chimericlight-activated proteins that do not have a mutation at E122. In someembodiments, the mutation can be a conservative amino acid substitution.In some embodiments, the mutation can be a non-conservative amino acidsubstitution. In some embodiments, the mutation at amino acid residueE122 can be to threonine (C1V1-E122T). In other embodiments, thelight-activated chimeric protein can comprise an amino acid sequence atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO: 3 without the signalpeptide sequence. In other embodiments, the light-activated chimericprotein can comprise an amino acid sequence at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown inSEQ ID NO: 3. In other embodiments, the C1V1-E122 mutant chimericlight-activated protein may be fused to a C-terminal trafficking signal.In some embodiments, the trafficking signal can be linked to theC1V1-E122 mutant chimeric light-activated protein by a linker. Thelinker can comprise any of 5, 10, 20, 30, 40, 50, 75, 100, 125, 150,175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. Thelinker may further comprise a fluorescent protein, for example, but notlimited to, an enhanced yellow fluorescent protein, a red fluorescentprotein, a green fluorescent protein, or a cyan fluorescent protein. Insome embodiments, the trafficking signal can be derived from the aminoacid sequence of the human inward rectifier potassium channel Kir2.1. Insome embodiments, the trafficking signal can comprise the amino acidsequence KSRITSEGEYIPLDQIDINV.

In other embodiments, the C1V1-E122 chimeric protein is capable ofmediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments the light can be greenlight. In other embodiments, the light can have a wavelength of betweenabout 540 nm to about 560 nm. In some embodiments, the light can have awavelength of about 546 nm. In other embodiments, the C1V1-E122 chimericprotein can mediate a depolarizing current in the cell when the cell isilluminated with red light. In some embodiments, the red light can havea wavelength of about 630 nm. In some embodiments, the C1V1-E122chimeric protein may not be capable of mediating a depolarizing currentin the cell when the cell is illuminated with violet light. In someembodiments, the chimeric protein may not be capable of mediating adepolarizing current in the cell when the cell is illuminated with lighthaving a wavelength of 405 nm. In some embodiments, the animal cell canbe a neuronal cell, a muscle cell, or a stem cell. In one embodiment,the animal cell can be a neuronal cell. In some embodiments the neuronalcell can be an excitatory neuron located in the pre-frontal cortex of anon-human animal. In other embodiments, the excitatory neuron can be apyramidal neuron. In some embodiments the neuronal cell can be aninhibitory neuron located in the pre-frontal cortex of a non-humananimal. In other embodiments, the excitatory neuron can be a pyramidalneuron. In still other embodiments, the inhibitory neuron can be aparvalbumin neuron. In some embodiments, the animal cells can furthercomprise a second light-activated protein expressed on the cells' plasmamembrane. In some embodiments, the second light-activated protein can becapable of mediating a hyperpolarizing current in the cell when the cellis illuminated with light. In some embodiments the secondlight-activated protein can be NpHr, eNpHr2.0, eNpHr3.0, eNpHr3.1 orGtR3.

C1V1-E162 Mutant Polypeptides

Provided herein are the light-activated C1V1 chimeric proteins disclosedherein expressed on an animal cell plasma membrane, wherein one or moreamino acid residues have undergone an amino acid substitution whileretaining C1V1 activity (i.e., the ability to catalyze thedepolarization of an animal cell in response to light activation),wherein the mutation can be at a glutamic acid residue corresponding toE162 of SEQ ID NO:1 (C1V1-E162). In some embodiments, the C1V1-E162mutant chimeric light-activated protein comprises substitutionsintroduced into the amino acid sequence shown in SEQ ID NO:1 at aminoacid E162 that can result in the chimeric protein having increasedsensitivity to light, increased sensitivity to particular wavelengths oflight, and/or increased ability to regulate the polarization state ofthe plasma membrane of the cell relative to C1V1 chimericlight-activated proteins that do not have a mutation at E162. In someembodiments, the mutation can be a conservative amino acid substitution.In some embodiments, the mutation can be a non-conservative amino acidsubstitution. In some embodiments, the mutation at amino acid residueE162 can be to threonine (C1V1-E162T). In other embodiments, thelight-activated chimeric protein can comprise an amino acid sequence atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO: 5 without the signalpeptide sequence. In other embodiments, the light-activated chimericprotein can comprise an amino acid sequence at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown inSEQ ID NO: 5. In other embodiments, the C1V1-E162 mutant chimericlight-activated protein may be fused to a C-terminal trafficking signal.In some embodiments, the trafficking signal can be linked to theC1V1-E162 mutant chimeric light-activated protein by a linker. Thelinker can comprise any of 5, 10, 20, 30, 40, 50, 75, 100, 125, 150,175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. Thelinker may further comprise a fluorescent protein, for example, but notlimited to, an enhanced yellow fluorescent protein, a red fluorescentprotein, a green fluorescent protein, or a cyan fluorescent protein. Insome embodiments, the trafficking signal can be derived from the aminoacid sequence of the human inward rectifier potassium channel Kir2.1. Insome embodiments, the trafficking signal can comprise the amino acidsequence KSRITSEGEYIPLDQIDINV.

In other embodiments, the C1V1-E162 chimeric protein is capable ofmediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments the light can be greenlight. In other embodiments, the light can have a wavelength of betweenabout 540 nm to about 535 nm. In some embodiments, the light can have awavelength of about 542 nm. In other embodiments, the light can have awavelength of about 530 nm. In some embodiments, the C1V1-E162 chimericprotein may not be capable of mediating a depolarizing current in thecell when the cell is illuminated with violet light. In someembodiments, the chimeric protein may not be capable of mediating adepolarizing current in the cell when the cell is illuminated with lighthaving a wavelength of 405 nm. In some embodiments, the C1V1-E162chimeric protein can further comprise a C-terminal fluorescent protein.In some embodiments, the animal cell can be a neuronal cell, a musclecell, or a stem cell. In one embodiment, the animal cell can be aneuronal cell. In some embodiments the neuronal cell can be anexcitatory neuron located in the pre-frontal cortex of a non-humananimal. In other embodiments, the excitatory neuron can be a pyramidalneuron. In some embodiments the neuronal cell can be an inhibitoryneuron located in the pre-frontal cortex of a non-human animal. In otherembodiments, the excitatory neuron can be a pyramidal neuron. In stillother embodiments, the inhibitory neuron can be a parvalbumin neuron. Insome embodiments, the animal cells can further comprise a secondlight-activated protein expressed on the cells' plasma membrane. In someembodiments, the second light-activated protein can be capable ofmediating a hyperpolarizing current in the cell when the cell isilluminated with light. In some embodiments the second light-activatedprotein can be NpHr, eNpHr2.0, eNpHr3.0, eNpHr3.1 or GtR3. In someembodiments, the C1V1-E162 light-activated chimeric protein can have anaccelerated photocycle relative C1V1 proteins lacking mutations at E162or relative to other light-activated cation channel proteins. In someembodiments, the C1V1-E162 light-activated chimeric protein can have aphotocycle more than 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5fold, 4 fold, 4.5 fold, or 5 fold faster than C1V1 proteins lackingmutations at E162 or relative to other light-activated cation channelproteins, inclusive.

C1V1-E122/E162 Double Mutant Polypeptides

Provided herein are the light-activated C1V1 chimeric proteins disclosedherein expressed on an animal cell plasma membrane, wherein one or moreamino acid residues have undergone an amino acid substitution whileretaining C1V1 activity (i.e., the ability to catalyze thedepolarization of an animal cell in response to light activation),wherein the mutations can be at glutamic acid residues corresponding toE122 and E162 of SEQ ID NO:1 (C1V1-E122/E162). In some embodiments, theC1V1-E122/E162 mutant chimeric light-activated protein can comprisesubstitutions introduced into the amino acid sequence shown in SEQ IDNO:1 at amino acid E122 and E162 that can result in the chimeric proteinhaving increased sensitivity to light, increased sensitivity toparticular wavelengths of light, and/or increased ability to regulatethe polarization state of the plasma membrane of the cell relative toC1V1 chimeric light-activated proteins that do not have a mutation atE122 and E162. In some embodiments, the mutations can be conservativeamino acid substitutions. In some embodiments, the mutations can benon-conservative amino acid substitutions. In some embodiments, themutations can be both conservative and non-conservative amino acidsubstitutions. In some embodiments, the mutation at amino acid residueE122 and at E162 can both be to threonine (C1V1-E122T/E162T). In otherembodiments, the light-activated chimeric protein can comprise an aminoacid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% identical to the sequence shown in SEQ ID NO: 7 without thesignal peptide sequence. In other embodiments, the light-activatedchimeric protein can comprise an amino acid sequence at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence shown in SEQ ID NO: 7. In other embodiments, the C1V1-E122/E162mutant chimeric light-activated protein may be fused to a C-terminaltrafficking signal. In some embodiments, the trafficking signal can belinked to the C1V1-E122/E162 mutant chimeric light-activated protein bya linker. The linker can comprise any of 5, 10, 20, 30, 40, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids inlength. The linker may further comprise a fluorescent protein, forexample, but not limited to, an enhanced yellow fluorescent protein, ared fluorescent protein, a green fluorescent protein, or a cyanfluorescent protein. In some embodiments, the trafficking signal can bederived from the amino acid sequence of the human inward rectifierpotassium channel Kir2.1. In some embodiments, the trafficking signalcan comprise the amino acid sequence KSRITSEGEYIPLDQIDINV.

In other embodiments, the C1V1-E122/E162 chimeric protein is capable ofmediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments the light can be greenlight. In other embodiments, the light can have a wavelength of betweenabout 540 nm to about 560 nm. In some embodiments, the light can have awavelength of about 546 nm. In some embodiments, the C1V1-E122/E162chimeric protein may not be capable of mediating a depolarizing currentin the cell when the cell is illuminated with violet light. In someembodiments, the chimeric protein may not be capable of mediating adepolarizing current in the cell when the cell is illuminated with lighthaving a wavelength of 405 nm. In some embodiments, the C1V1-E122/E162chimeric protein can exhibit less activation when exposed to violetlight relative to C1V1 proteins lacking mutations at E122/E162 orrelative to other light-activated cation channel proteins. In someembodiments, the animal cell can be a neuronal cell, a muscle cell, or astem cell. In one embodiment, the animal cell can be a neuronal cell. Insome embodiments the neuronal cell can be an excitatory neuron locatedin the pre-frontal cortex of a non-human animal. In other embodiments,the excitatory neuron can be a pyramidal neuron. In some embodiments theneuronal cell can be an inhibitory neuron located in the pre-frontalcortex of a non-human animal. In still other embodiments, the inhibitoryneuron can be a parvalbumin neuron. In some embodiments, the animalcells can further comprise a second light-activated protein expressed onthe cells' plasma membrane. In some embodiments, the secondlight-activated protein can be capable of mediating a hyperpolarizingcurrent in the cell when the cell is illuminated with light. In someembodiments the second light-activated protein can be NpHr, eNpHr2.0,eNpHr3.0, eNpHr3.1 or GtR3. In some embodiments, the C1V1-E122/E162mutant light-activated chimeric protein can have decreased inactivationrelative to C1V1 proteins lacking mutations at E122/E162 or relative toother light-activated cation channel proteins. In some embodiments, theC1V1-E122/E162 mutant light-activated chimeric protein can inactivate byany of about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26v,27%, 28%, 29%, or 30% compared to C1V1 proteins lacking mutations atE122/E162 or relative to other light-activated cation channel proteins,inclusive. In some embodiments, the C1V1-E122/E162 light-activatedchimeric protein can have a photocycle more than 1 fold, 1.5 fold, 2fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, or10 fold faster than C1V1 proteins lacking mutations at E122/E162 orrelative to other light-activated cation channel proteins, inclusive.

Enhanced Intracellular Transport Amino Acid Motifs

The present disclosure provides for the modification of light-activatedchimeric proteins expressed in a cell by the addition of one or moreamino acid sequence motifs which enhance transport to the plasmamembranes of mammalian cells. Light-activated chimeric proteins havingcomponents derived from evolutionarily simpler organisms may not beexpressed or tolerated by mammalian cells or may exhibit impairedsubcellular localization when expressed at high levels in mammaliancells. Consequently, in some embodiments, the chimeric light-activatedprotein expressed in a cell is fused to one or more amino acid sequencemotifs selected from the group consisting of a signal peptide, anendoplasmic reticulum (ER) export signal, a membrane trafficking signal,and an N-terminal golgi export signal. The one or more amino acidsequence motifs which enhance light-activated chimeric protein transportto the plasma membranes of mammalian cells can be fused to theN-terminus, the C-terminus, or to both the N- and C-terminal ends of thelight-activated protein. Optionally, the light-activated protein and theone or more amino acid sequence motifs may be separated by a linker. Insome embodiments, the light-activated chimeric protein is modified bythe addition of a trafficking signal (ts) which enhances transport ofthe protein to the cell plasma membrane. In some embodiments, thetrafficking signal is derived from the amino acid sequence of the humaninward rectifier potassium channel Kir2.1. In some embodiments, thetrafficking signal comprises the amino acid sequenceKSRITSEGEYIPLDQIDINV. Additional protein motifs which can enhancelight-activated protein transport to the plasma membrane of a cell aredescribed in U.S. patent application Ser. No. 12/041,628, which isincorporated herein by reference in its entirety. In some embodiments,the signal peptide sequence in the chimeric protein is deleted orsubstituted with a signal peptide sequence from a different protein.

Animal Cells, Non-Human Animals, and Brain Slices

Provided herein are cells comprising the light activated chimericproteins disclosed herein. In some embodiments, the cells are animalcells. In some embodiments, the animal cells comprise the C1V1 proteincorresponding to SEQ ID NO:1. In other embodiments, the animal cellscomprise the mutant C1V1-E122T protein corresponding to SEQ ID NO:3. Inother embodiments, the animal cells comprise the mutant C1V1-E162Tprotein corresponding to SEQ ID NO:5. In other embodiments, the animalcells comprise the mutant C1V1-E122T/E162T protein corresponding to SEQID N07. In some embodiments, the animal cell can be a neuronal cell, amuscle cell, or a stem cell. In one embodiment, the animal cell can be aneuronal cell. In some embodiments the neuronal cell can be anexcitatory neuron located in the pre-frontal cortex of a non-humananimal. In other embodiments, the excitatory neuron can be a pyramidalneuron. In some embodiments the neuronal cell can be an inhibitoryneuron located in the pre-frontal cortex of a non-human animal. In stillother embodiments, the inhibitory neuron can be a parvalbumin neuron.

Also provided herein, are non-human animals comprising the lightactivated chimeric proteins disclosed herein expressed on the cellmembrane of the cells in the animals. In some embodiments, the animalcells comprise the C1V1 protein corresponding to SEQ ID NO:1. In otherembodiments, the animal cells comprise the mutant C1V1-E122T proteincorresponding to SEQ ID NO:3. In other embodiments, the animal cellscomprise the mutant C1V1-E162T protein corresponding to SEQ ID NO:5. Inother embodiments, the animal cells comprise the mutant C1V1-E122T/E162Tprotein corresponding to SEQ ID N07. In some embodiments, the animalscomprising the light-activated chimeric proteins described herein aretransgenically expressing said light-activated chimeric proteins. Inother embodiments, the animals comprising the light-activated chimericproteins described herein have been virally transfected with a vectorcarrying the light-activated protein such as, but not limited to, anadenoviral vector.

Provided herein are living brain slices from a non-human animalcomprising the light-activated chimeric proteins described hereinexpressed on the cell membrane of the cells in the slices. In someembodiments, the brain slices are from non-human animals transgenicallyexpressing the light-activated chimeric proteins described herein. Inother embodiments, the brain slices are from non-human animals that havebeen virally transfected with a vector carrying said light-activatedprotein such as, but not limited to, an adenoviral vector. In someembodiments, the brain slices are coronal brain slices. In someembodiments, the brain slices are any of about 100 μm, about 150 μm,about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm,about 450 μm, or about 500 μm thick, inclusive, including anythicknesses in between these numbers.

Isolated Polynucleotides

Provided herein are isolated C1V1 polynucleotides that encode anychimeric polypeptides described herein that, for example, have at leastone activity of a C1V1 polypeptide. The disclosure provides isolated,synthetic, or recombinant polynucleotides comprising a nucleic acidsequence having at least about 70%, e.g., at least about 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%; 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, orcomplete (100%) sequence identity to the nucleic acid of SEQ ID NO:2, 4,6 or 8 over a region of at least about 10, e.g., at least about 15, 20,25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides.

The disclosure specifically provides a nucleic acid encoding C1V1 and/ora mutant variant thereof. For example, the disclosure provides anisolated nucleic acid molecule, wherein the nucleic acid moleculeencodes: (1) a polypeptide comprising an amino acid sequence with atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:1; (2) a polypeptidecomprising an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acidsequence of SEQ ID NO:3, (3) a polypeptide comprising an amino acidsequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity to the amino acid sequence of SEQ ID NO:5; or(4) a polypeptide comprising an amino acid sequence with at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity tothe amino acid sequence represented by SEQ ID NO:7.

Promoters and Vectors

The disclosure also provides expression cassettes and/or vectorscomprising the above-described nucleic acids. Suitably, the nucleic acidencoding a chimeric protein of the disclosure is operably linked to apromoter. Promoters are well known in the art. Any promoter thatfunctions in the host cell can be used for expression of C1V1 and/or anyvariant thereof of the present disclosure. Initiation control regions orpromoters, which are useful to drive expression of a C1V1 chimericprotein or variant thereof in a specific animal cell are numerous andfamiliar to those skilled in the art. Virtually any promoter capable ofdriving these nucleic acids can be used.

Specifically, where recombinant expression of C1V1 chimeric proteins inan excitatory neural cell is desired, a human calmodulin-dependentprotein kinase II alpha (CaMKIIα) promoter may be used. In otherembodiments, an elongation factor 1a (EF-1a) promoter in conjunctionwith a Cre-inducible recombinant AAV vector can be used withparvalbumin-Cre transgenic mice to target expression C1V1 chimericproteins to inhibitory neurons.

Also provided herein are vectors comprising the polynucleotidesdisclosed herein encoding a C1V1 chimeric polypeptide or any variantthereof. The vectors that can be administered according to the presentinvention also include vectors comprising a polynucleotide which encodesan RNA (e.g., RNAi, ribozymes, miRNA, siRNA) that when transcribed fromthe polynucleotides of the vector will result in the accumulation oflight-activated chimeric proteins on the plasma membranes of targetanimal cells. Vectors which may be used, include, without limitation,lentiviral, HSV, and adenoviral vectors. Lentiviruses include, but arenot limited to HIV-1, HIV-2, SIV, FIV and EIAV. Lentiviruses may bepseudotyped with the envelope proteins of other viruses, including, butnot limited to VSV, rabies, Mo-MLV, baculovirus and Ebola. Such vectorsmay be prepared using standard methods in the art.

In some embodiments, the vector is a recombinant AAV vector. AAV vectorsare DNA viruses of relatively small size that can integrate, in a stableand sitespecific manner, into the genome of the cells that they infect.They are able to infect a wide spectrum of cells without inducing anyeffects on cellular growth, morphology or differentiation, and they donot appear to be involved in human pathologies. The AAV genome has beencloned, sequenced and characterized. It encompasses approximately 4700bases and contains an inverted terminal repeat (ITR) region ofapproximately 145 bases at each end, which serves as an origin ofreplication for the virus. The remainder of the genome is divided intotwo essential regions that carry the encapsidation functions: theleft-hand part of the genome, that contains the rep gene involved inviral replication and expression of the viral genes; and the right-handpart of the genome, that contains the cap gene encoding the capsidproteins of the virus.

The application of AAV as a vector for gene therapy has been rapidlydeveloped in recent years. Wild-type AAV could infect, with acomparatively high titer, dividing or non-dividing cells, or tissues ofmammal, including human, and also can integrate into in human cells atspecific site (on the long arm of chromosome 19) (Kotin, R. M., et al,Proc. Natl. Acad. Sci. USA 87: 2211-2215, 1990) (Samulski, R. J, et al,EMBO J. 10: 3941-3950, 1991 the disclosures of which are herebyincorporated by reference herein in their entireties). AAV vectorwithout the rep and cap genes loses specificity of site-specificintegration, but may still mediate long-term stable expression ofexogenous genes. AAV vector exists in cells in two forms, wherein one isepisomic outside of the chromosome; another is integrated into thechromosome, with the former as the major form. Moreover, AAV has nothitherto been found to be associated with any human disease, nor anychange of biological characteristics arising from the integration hasbeen observed. There are sixteen serotypes of AAV reported inliterature, respectively named AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, whereinAAV5 is originally isolated from humans (Bantel-Schaal, and H. zurHausen. 1984. Virology 134: 52-63), while AAV1-4 and AAV6 are all foundin the study of adenovirus (Ursula Bantel-Schaal, Hajo Delius and Haraldzur Hausen. J. Virol. 1999, 73: 939-947).

AAV vectors may be prepared using standard methods in the art.Adeno-associated viruses of any serotype are suitable (See, e.g.,Blacklow, pp. 165-174 of “Parvoviruses and Human Disease” J. R.Pattison, ed. (1988); Rose, Comprehensive Virology 3:1, 1974; P.Tattersall “The Evolution of Parvovirus Taxonomy” In Parvoviruses (J RKerr, S F Cotmore. M E Bloom, R M Linden, C R Parrish, Eds.) p 5-14,Hudder Arnold, London, UK (2006); and D E Bowles, J E Rabinowitz, R JSamulski “The Genus Dependovirus” (J R Kerr, S F Cotmore. M E Bloom, R MLinden, C R Parrish, Eds.) p 15-23, Hudder Arnold, London, UK (2006),the disclosures of which are hereby incorporated by reference herein intheir entireties). Methods for purifying for vectors may be found in,for example, U.S. Pat. Nos. 6,566,118, 6,989,264, and 6,995,006 andWO/1999/011764 titled “Methods for Generating High Titer Helper-freePreparation of Recombinant AAV Vectors”, the disclosures of which areherein incorporated by reference in their entirety. Preparation ofhybrid vectors is described in, for example, PCT Application No.PCT/US2005/027091, the disclosure of which is herein incorporated byreference in its entirety. The use of vectors derived from the AAVs fortransferring genes in vitro and in vivo has been described (See e.g.,International Patent Application Publication Nos: 91/18088 and WO93/09239; U.S. Pat. Nos. 4,797,368, 6,596,535, and 5,139,941; andEuropean Patent No: 0488528, all of which are herein incorporated byreference in their entirety). These publications describe variousAAV-derived constructs in which the rep and/or cap genes are deleted andreplaced by a gene of interest, and the use of these constructs fortransferring the gene of interest in vitro (into cultured cells) or invivo (directly into an organism). The replication defective recombinantAAVs according to the invention can be prepared by co-transfecting aplasmid containing the nucleic acid sequence of interest flanked by twoAAV inverted terminal repeat (ITR) regions, and a plasmid carrying theAAV encapsidation genes (rep and cap genes), into a cell line that isinfected with a human helper virus (for example an adenovirus). The AAVrecombinants that are produced are then purified by standard techniques.

In some embodiments, the vector(s) for use in the methods of theinvention are encapsidated into a virus particle (e.g. AAV virusparticle including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, andAAV16). Accordingly, the invention includes a recombinant virus particle(recombinant because it contains a recombinant polynucleotide)comprising any of the vectors described herein. Methods of producingsuch particles are known in the art and are described in U.S. Pat. No.6,596,535.

For the animal cells described herein, it is understood that one or morevectors may be administered to neural cells, heart cells, or stem cells.If more than one vector is used, it is understood that they may beadministered at the same or at different times to the animal cell.

Methods of the Invention

Provided herein are methods for selectively depolarizing excitatory orinhibitory neurons residing in the same microcircuit by expressing inthose neurons the light-activated chimeric proteins described herein. Insome embodiments, a first light-activated protein, such as thosedisclosed herein, can be expressed in an excitatory neuron while asecond light-activated protein can be expressed in an inhibitory neuron.In some embodiments, the first light-activated protein expressed in theexcitatory neuron can be activated by a different wavelength of lightthan the second light-activated protein expressed in the inhibitoryneuron. In some embodiments, the first and second light-activatedproteins can be expressed in a living non-human animal or in a livingbrain slice from a non-human animal.

In other embodiments, a method is provided for identifying a chemicalcompound that selectively inhibits the depolarization of excitatory orinhibitory neurons residing in the same neural circuit by expressing inthose neurons the light-activated chimeric proteins described herein. Insome embodiments, a first light-activated protein can be expressed in anexcitatory neuron while a second light-activated protein can beexpressed in an inhibitory neuron. In some embodiments, the firstlight-activated protein expressed in the excitatory neuron can beactivated by a different wavelength of light than the secondlight-activated protein expressed in the inhibitory neuron. In someembodiments, the first and second light-activated proteins can beexpressed in a living non-human animal or in a living brain slice from anon-human animal.

Methods for Selectively Altering the Eli Balance in Neurons Residing inthe Same Microcircuit

In some aspects, there is provided a method for selectively depolarizingexcitatory or inhibitory neurons residing in the same microcircuit, themethod comprising: selectively depolarizing an excitatory neuroncomprising a first light-activated protein, wherein the firstlight-activated protein is depolarized when exposed to light having afirst wavelength or selectively depolarizing an inhibitory neuroncomprising a second light-activated protein, wherein the secondlight-activated protein is depolarized when exposed to light having asecond wavelength. In some embodiments, the first light-activatedprotein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown inSEQ ID NO: 1. In other embodiments, the first light-activated proteincan comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence shown in SEQ IDNO: 3. In some embodiments, the first light-activated protein cancomprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 5.In some embodiments, the second light-activated protein can comprise aprotein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence shown in SEQ ID NO:11. In someembodiments, the second light-activated protein can comprise a proteinat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence shown in SEQ ID NO:12. In someembodiments, the second light-activated protein can comprise a proteinat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence shown in SEQ ID NO: 13. In someembodiments, the second light-activated protein can comprise a proteinat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence shown in SEQ ID NO: 14. Moreinformation regarding the disclosure of other light-activated cationchannels can be found in U.S. Patent Application Publication No:2007/0054319; U.S. Patent Application No. 61/410,704; and InternationalPatent Application Publication No: WO 2010/056970, the disclosures ofeach of which are hereby incorporated by reference in their entireties.

In other aspects, there is provided a method for selectivelydepolarizing excitatory or inhibitory neurons residing in the samemicrocircuit, the method comprising: expressing a first light-activatedprotein in an excitatory neuron; and expressing a second light-activatedprotein in an inhibitory neuron, wherein the first light-activatedprotein is independently depolarized when exposed to light having afirst wavelength and wherein the second light-activated protein isindependently depolarized when exposed to light having a secondwavelength. In some embodiments, the first light-activated protein cancomprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 1.In other embodiments, the first light-activated protein can comprise aprotein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence shown in SEQ ID NO: 3. In someembodiments, the first light-activated protein can comprise a protein atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identicalto the amino acid sequence shown in SEQ ID NO: 5. In some embodiments,the second light-activated protein can comprise a protein at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence shown in SEQ ID NO:11. In some embodiments, thesecond light-activated protein can comprise a protein at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence shown in SEQ ID NO:12. In some embodiments, the secondlight-activated protein can comprise a protein at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence shown in SEQ ID NO:13. In some embodiments, the secondlight-activated protein can comprise a protein at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence shown in SEQ ID NO:14.

In some embodiments, the first light-activated protein can be activatedby green light. In one embodiment, the first light-activated protein canbe activated by light having a wavelength of about 560 nm. In oneembodiment, the first light-activated protein can be activated by redlight. In another embodiment, the first light-activated protein can beactivated by light having a wavelength of about 630 nm. In otherembodiments, the second light-activated protein can be activated byviolet light. In one embodiment, the second light-activated protein canbe activated by light having a wavelength of about 405 nm. In otherembodiments, the second light activated protein can be activated bygreen light. In some embodiments, the light-activated proteins areactivated by light pulses that can have a duration for any of about 1millisecond (ms), about 2 ms, about 3, ms, about 4, ms, about 5 ms,about 6 ms, about 7 ms, about 8 ms, about 9 ms, about 10 ms, about 15ms, about 20 ms, about 25 ms, about 30 ms, about 35 ms, about 40 ms,about 45 ms, about 50 ms, about 60 ms, about 70 ms, about 80 ms, about90 ms, about 100 ms, about 200 ms, about 300 ms, about 400 ms, about 500ms, about 600 ms, about 700 ms, about 800 ms, about 900 ms, about 1 sec,about 1.25 sec, about 1.5 sec, or about 2 sec, inclusive, including anytimes in between these numbers. In some embodiments, the light-activatedproteins are activated by light pulses that can have a light powerdensity of any of about 0.05 mW mm⁻², about 0.1 mW mm², about 0.25 mWmm⁻², about 0.5 mW mm⁻², about 0.75 mW mm⁻², about 1 mW mm⁻², about 2 mWmm⁻², about 3 mW mm⁻², about 4 mW mm⁻², about 5 mW mm⁻², about 6 mWmm⁻², about 7 mW mm⁻², about 8 mW mm⁻², about 9 mW mm⁻², about 10 mWmm⁻², about 11 mW mm⁻², about 12 mW mm⁻², about 13 mW mm⁻², about 14 mWmm⁻², about mW mm⁻², about 16 mW mm⁻², about 17 mW mm⁻², about 18 mWmm⁻², about 19 mW mm⁻², about 20 mW mm⁻², about 21 mW mm⁻², about 22 mWmm⁻², about 23 mW mm⁻², about 24 mW mm⁻², or about 25 mW mm⁻²,inclusive, including any values between these numbers. In someembodiments the neuronal cell can be an excitatory neuron located in thepre-frontal cortex of a non-human animal. In other embodiments, theexcitatory neuron can be a pyramidal neuron. In some embodiments theneuronal cell can be an inhibitory neuron located in the pre-frontalcortex of a non-human animal. In still other embodiments, the inhibitoryneuron can be a parvalbumin neuron. In some embodiments, the inhibitoryand excitatory neurons can be in a living non-human animal. In otherembodiments, the inhibitory and excitatory neurons can be in a brainslice from a non-human animal.

Methods for Identifying a Chemical Compound that Selectively Alters theE/I Balance in Neurons Residing in the Same Microcircuit

In some aspects, there is provided a method for identifying a chemicalcompound that selectively inhibits the depolarization of excitatory orinhibitory neurons residing in the same microcircuit, the methodcomprising: (a) selectively depolarizing an excitatory neuron comprisinga first light-activated protein with light having a first wavelength orselectively depolarizing an inhibitory neuron comprising a secondlight-activated protein with light having a second wavelength; (b)measuring an excitatory post synaptic potential (EPSP) in response toselectively depolarizing the excitatory neuron comprising a firstlight-activated protein or measuring an inhibitory post synaptic current(IPSC) in response to selectively depolarizing an inhibitory neuroncomprising a second light-activated protein; (c) contacting theexcitatory neuron or the inhibitory neuron with a chemical compound; (d)measuring the excitatory post synaptic potential (EPSP) or measuring theinhibitory post synaptic current (IPSC) to determine if contactingeither the excitatory neuron or the inhibitory neuron with the chemicalcompound selectively inhibits the depolarization of either neuron. Insome embodiments, the first light-activated protein can comprise aprotein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence shown in SEQ ID NO: 1. Inother embodiments, the first light-activated protein can comprise aprotein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence shown in SEQ ID NO: 3. In someembodiments, the first light-activated protein can comprise a protein atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identicalto the amino acid sequence shown in SEQ ID NO: 5. In some aspects, thesecond light-activated protein can comprise a protein at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence shown in SEQ ID NO:11. In some embodiments, the secondlight-activated protein can comprise a protein at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence shown in SEQ ID NO:12. In some embodiments, the secondlight-activated protein can comprise a protein at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence shown in SEQ ID NO:13. In some embodiments, the secondlight-activated protein can comprise a protein at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence shown in SEQ ID NO:14. In some embodiments, the chemicalcompound can be a member of a combinatorial chemical library. In otherembodiments, the method further comprises assaying the chemical compoundto determine if it adversely affects the function of cardiac tissue orthe cardiac action potential in mammals.

In some embodiments, the first light-activated protein can be activatedby green light. In one embodiment, the first light-activated protein canbe activated by light having a wavelength of about 560 nm. In oneembodiment, the first light-activated protein can be activated by redlight. In another embodiment, the first light-activated protein can beactivated by light having a wavelength of about 630 nm. In otherembodiments, the second light-activated protein can be activated byviolet light. In one embodiment, the second light-activated protein canbe activated by light having a wavelength of about 405 nm. In someembodiments, the light-activated proteins can be activated by lightpulses that can have a duration for any of about 1 millisecond (ms),about 2 ms, about 3, ms, about 4, ms, about 5 ms, about 6 ms, about 7ms, about 8 ms, about 9 ms, about 10 ms, about 15 ms, about 20 ms, about25 ms, about 30 ms, about 35 ms, about 40 ms, about 45 ms, about 50 ms,about 60 ms, about 70 ms, about 80 ms, about 90 ms, about 100 ms, about200 ms, about 300 ms, about 400 ms, about 500 ms, about 600 ms, about700 ms, about 800 ms, about 900 ms, about 1 sec, about 1.25 sec, about1.5 sec, or about 2 sec, inclusive, including any times in between thesenumbers. In some embodiments, the light-activated proteins can beactivated by light pulses that can have a light power density of any ofabout 0.05 mW mm⁻², about 0.1 mW mm⁻², about 0.25 mW mm⁻², about 0.5 mWmm⁻², about 0.75 mW mm⁻², about 1 mW mm⁻², about 2 mW mm⁻², about 3 mWmm⁻², about 4 mW mm⁻², about 5 mW mm⁻², about 6 mW mm⁻², about 7 mWmm⁻², about 8 mW mm⁻², about 9 mW mm⁻², about 10 mW mm⁻², about 11 mWmm⁻², about 12 mW mm⁻², about 13 mW mm⁻², about 14 mW mm⁻², about mWmm⁻², about 16 mW mm⁻², about 17 mW mm⁻², about 18 mW mm⁻², about 19 mWmm⁻², about 20 mW mm⁻², about 21 mW mm⁻², about 22 mW mm⁻², about 23 mWmm⁻², about 24 mW mm⁻², or about 25 mW mm⁻², inclusive, including anyvalues between these numbers. In some embodiments the neuronal cell canbe an excitatory neuron located in the pre-frontal cortex of a non-humananimal. In other embodiments, the excitatory neuron can be a pyramidalneuron. In some embodiments the neuronal cell can be an inhibitoryneuron located in the pre-frontal cortex of a non-human animal. In stillother embodiments, the inhibitory neuron can be a parvalbumin neuron. Insome embodiments, the inhibitory and excitatory neurons can be in aliving non-human animal. In other embodiments, the inhibitory andexcitatory neurons can be in a brain slice from a non-human animal.

Exemplary Embodiments

The present disclosure relates to a light-activated chimera opsin thatmodifies a membrane voltage when expressed therein. While the presentdisclosure is not necessarily limited in these contexts, various aspectsof the disclosure may be appreciated through a discussion of examplesusing these and other contexts.

Various embodiments of the present disclosure relate to alight-activated opsin modified for expression in cell membranesincluding mammalian cells. The opsin is derived from a combination oftwo different opsins, Volvox channelrhodopsin (VChR1) and Chlamydomonasreinhardtii channelrhodopsin (ChR1). The opsin can be useful forexpressing at levels of a higher rate than either of the individualopsins from which it is derived.

In certain more specific embodiments, the genetic sequence of ChR1/VChR1chimera (C1V1) is primarily VChR1. Portions of the VChR1 sequenceassociated with trafficking are replaced with homologous sequences fromChR1.

Various embodiments relate to modification directed toward the additionof a trafficking signal to improve expression in mammalian cells.

Certain aspects of the present disclosure are directed to furthermodified versions of C1V1. For example, certain embodiments include amutation E162T to C1V1, which experiments suggest provides anaccelerated photocycle (e.g., almost 3-fold). Various embodiments of thepresent disclosure relate to an optogenetic system or method thatcorrelates temporal, spatial and/or cell-type-specific control over aneural circuit with measurable metrics. The optogenetic system uses avariety of opsins, including C1V1 and/or C1V1 variants, to assertcontrol over portions of neural circuits. For instance, various metricsor symptoms might be associated with a neurological disorder. Theoptogenetic system targets a neural circuit within a patient forselective control thereof. The optogenetic system involves monitoringthe patient for the metrics or symptoms associated with the neurologicaldisorder. In this manner, the optogenetic system can provide detailedinformation about the neural circuit, its function and/or theneurological disorder.

Consistent with the embodiments discussed herein, particular embodimentsrelate to studying and probing disorders using a variety of opsins.Other embodiments relate to the identification and/or study ofphenotypes and endophenotypes. Still other embodiments relate to theidentification of treatment targets.

Aspects of the present disclosure are directed toward the artificialinducement of disorder/disease states on a fast-temporal time scale. Theuse of an opsin such as C1V1 can be particularly useful based oncharacteristics regarding an accelerated photocycle. Moreover, certainembodiments allow for reversible disease states, which can beparticularly useful for establishing baseline/control points for testingand/or for testing the effects of a treatment on the same animal whenexhibiting the disease state and when not exhibiting the disease state.The use of opsins such as C1V1 allows for the control of a cell using alight source. The C1V1 reacts to light, causing a change in the membranepotential of the cell. The removal of the light and the subsequentcessation of the activation of C1V1 allows for the cell to return to itsbaseline state. Various other possibilities exist, some of which arediscussed in more detail herein.

Various aspects of the present disclosure are directed to an E122Tmutation of a C1V1 opsin. In certain embodiments of the presentdisclosure, the E122T mutation shifts maximum absorption of C1V1 or itsvariants toward the red light spectrum with respect to the un-mutatedopsin.

Various embodiments of the present disclosure relate to an opsinmodified for expression in mammalian cells and shifted, with respect toChR2, for maximum absorption in the green light spectrum. The C1V1 opsinis derived from a combination of opsins and expresses at a higher ratethan either of the opsins from which it is derived. The opsin, C1V1, isderived from Volvox channelrhodopsin (VChR1) and Chlamydomonasreinhardtii channelrhodopsin (ChR1). The resulting opsin, C1V1 and itsvariants, have a maximum absorption at wavelengths between 530 nm and546 nm.

Certain aspects of the present disclosure are directed to furthermodified versions of C1V1. For example, certain embodiments include amutation E122T, which shifts the maximum absorption of C1V1 towards thered light spectrum. Other modifications can include an additionalmutation E162T, which experiments suggest provides an acceleratedphotocycle in addition to the red shift provided by the E122T mutation.

In some embodiments, there is provided a transmembrane molecule derivedfrom VChR1 and having the traffic sequences replaced with homologoussequences from ChR1. In some embodiments, the molecule further includesa mutation E122T. In other embodiments, the molecule further includesmutations at E162T and E122T. In certain embodiments, the moleculeactivates an ion channel in response to green light. In one embodiment,the molecule has a maximum light absorption of approximately 546 nm. Inanother embodiment, the molecule has a maximum light absorption ofapproximately 535 nm.

In some embodiments, there is provided an animal cell comprising: anintegrated exogenous molecule which expresses an ion channel that isresponsive to red light; the exogenous molecule derived from VChR1 andincluding transmembrane traffic sequences thereof replaced by homologoussequences from ChR1. In some embodiments, the exogenous molecule furtherincludes E122T. In other embodiments, the cell has a neural firing ratioof about 14% to 94% in response to light having wavelengths of 405 nmand 560 nm, respectively. In other embodiments, the cell has a neuralfiring ratio of about 11% to 72% in response to light having wavelengthsof 405 nm and 560 nm, respectively.

Additional example embodiments of the present disclosure relate to theuse of a hybrid ChR1/VChR1 chimera that contains no ChR2 sequence atall, is derived from two opsins genes that do not express wellindividually, and is herein referred to as C1V1. Embodiments of thepresent disclosure also relate to improvements of the membrane targetingof VChR1 through the addition of a membrane trafficking signal derivedfrom the K_(ir)2.1 channel. Confocal images from cultured neuronsexpressing VChR1-EYFP revealed a large proportion of intracellularprotein compared with ChR2; therefore, membrane trafficking signal (ts)derived from the K_(ir)2.1 channel was used to improve the membranetargeting of VChR1. Membrane targeting of this VChR1-ts-EYFP wasslightly enhanced compared with VChR1-EYFP; however, mean photocurrentsrecorded from cultured hippocampal neurons expressing VChR1-ts-EYFP wereonly slightly larger than those of VChR1-EYFP. Accordingly, embodimentsof the present disclosure relate to VChR1, which has been modified byexchanging helices with corresponding helices from other ChRs. Forexample, robust improvement has been discovered in two chimeras wherehelices 1 and 2 were replaced with the homologous segments from ChR1. Itwas discovered that whether splice sites were in the intracellular loopbetween helices 2 and 3 (at ChR1 residue Ala145) or within helix 3 (atChR1 residue Trp163), the resulting chimeras were both robustlyexpressed and showed similarly enhanced photocurrent and spectralproperties. This result was unexpected as ChR1 is only weakly expressedand poorly integrated into membranes of most mammalian host cells.

Specific aspects of the present disclosure relate to microbial opsingenes adapted for neuroscience, allowing transduction of light pulsetrains into millisecond-timescale membrane potential changes in specificcell types within the intact mammalian brain (e.g., channelrhodopsin(ChR2), Volvox channelrhodopsin (VChR1) and halorhodopsin (NpHR)). ChR2is a rhodopsin derived from the unicellular green algae Chlamydomonasreinhardtii. The term “rhodopsin” as used herein is a protein thatcomprises at least two building blocks, an opsin protein, and acovalently bound cofactor, usually retinal (retinaldehyde). Therhodopsin ChR2 is derived from the opsin Channelopsin-2 (Chop2),originally named Chlamyopsin-4 (Cop4) in the Chlamydomonas genome. Thetemporal properties of one depolarizing channelrhodopsin, ChR2, includefast kinetics of activation and deactivation, affording generation ofprecisely timed action potential trains. For applications seeking longtimescale activation, it has been discovered that the normally fastoff-kinetics of the channelrhodopsins can be slowed. For example,certain implementations of channelrhodopsins apply 1 mW/mm² light forvirtually the entire time in which depolarization is desired, which canbe less than desirable.

Much of the discussion herein is directed to ChR2. Unless otherwisestated, the disclosure includes a number of similar variants. Examplesinclude, but are not limited to, Chop2, ChR2-310, Chop2-310, and Volvoxchannelrhodopsin (VChR1). For further details on VChR1, reference can bemade to “Red-shifted optogenetic excitation: a tool for fast neuralcontrol derived from Volvox carteri,” Nat Neurosci. June 2008,11(6):631-3. Epub 2008 Apr. 23, the disclosure of which is fullyincorporated herein by reference in its entirety. In otherimplementations, similar modifications can be made to other opsinmolecules. For instance, modifications/mutations can be made to ChR2 orVChR1 variants. Moreover the modified variants can be used incombination with light-activated ion pumps.

Embodiments of the present disclosure include relatively minor aminoacid variants of the naturally occurring sequences. In one instance, thevariants are greater than about 75% homologous to the protein sequenceof the naturally occurring sequences. In other variants, the homology isgreater than about 80%. Yet other variants have homology greater thanabout 85%, greater than 90%, or even as high as about 93% to about 95%or about 98%. Homology in this context means sequence similarity oridentity, with identity being preferred. This homology can be determinedusing standard techniques known in sequence analysis. The compositionsof embodiments of the present disclosure include the protein and nucleicacid sequences provided herein, including variants which are more thanabout 50% homologous to the provided sequence, more than about 55%homologous to the provided sequence, more than about 60% homologous tothe provided sequence, more than about 65% homologous to the providedsequence, more than about 70% homologous to the provided sequence, morethan about 75% homologous to the provided sequence, more than about 80%homologous to the provided sequence, more than about 85% homologous tothe provided sequence, more than about 90% homologous to the providedsequence, or more than about 95% homologous to the provided sequence.

As used herein, “stimulation of a target cell” is generally used todescribe modification of the properties of the cell. For instance, thestimulus of a target cell may result in a change in the properties ofthe cell membrane that can lead to the depolarization or polarization ofthe target cell. In a particular instance, the target cell is a neuronand the stimulus affects the transmission of impulses by facilitating orinhibiting the generation of impulses (action potentials) by the neuron.

For further details on light-activated opsins, reference can be made toPCT publication No. WO 2010/056970, entitled “Optically-BasedStimulation of Target Cells and Modifications Thereto,” to Deisseroth etal., which is fully incorporated herein by reference in its entirety.

EXAMPLES Example 1 Development of Chimeric Channelrhodopsin Variant C1V1

In this example, a tool that would permit the driving of cortical E/Ielevations and the monitoring of gamma oscillations in cortical slices,as well as in vivo in live animal experiments, was sought, with threekey properties: 1) much higher potency to enable dose-responseinvestigation; 2) low desensitization to allow for step-like changes inE/I balance; and 3) redshifted excitation to allow comparative drive ofdifferent populations within the same preparation.

These experiments were initially attempted with VChR1, which displaysboth a redshift and reduced desensitization¹⁴, but previousinvestigation suggested that photocurrents in cells expressing VChR1were small (−100−150 pA¹⁴), and did not elicit robust synaptic activityin downstream cells (not shown). Indeed, when first attempting toexpress VChR1 in cells, only small photocurrents were observed, (FIG. 1)consistent with previous findings. Adding a membrane trafficking signalderived from the Kir2.1 channel to generate VChR1-is-EYFP delivered onlya modest trend toward enhanced photocurrents compared with VChR1-EYFP(FIG. 2). However, noting that in ChR2, replacing transmembrane segmentswith the homologous region from ChR1 increased membrane targeting andenhanced photocurrents, it was hypothesized that a similar systematicexchange between the helices of VChR1 with the corresponding helicesfrom other ChRs, might similarly result in enhanced membrane expressionin HEK cells.

Materials and Methods

Chimeric channelrhodopsin variant C1V1 was generated by fusing either awild-type or human codon-optimized channelrhodopsin-1 with a humancodon-adapted VChR1 (GenBank™ accession number ACD70142.1) by overlapextension PCR. C1V1 splice variants were generated by overlap PCR.Variant one contained the first 145 amino acids of ChR1 and amino acids102 to 316 of VChR1. Variant two contained the first 162 amino acids ofChR1 and amino acids 119 to 316 of VChR1. The resultant chimeric PCRfragments were cloned into pECFP-N1 (Clonetech, Mountain View, Calif.)and into lentiviral expression vectors under the CaMKIIα promoter. Themembrane trafficking signal was derived from the Kir2.1 channel.Mutations were confirmed by sequencing the coding sequence and splicesites. For AAV-mediated gene delivery, opsin-EYFP fusions along with theCaMKIIα promoter were subcloned into a modified version of the pAAV2-MCSvector. Cre-dependent opsin expression was achieved by cloning theopsin-EYFP cassette in the reverse orientation between pairs ofincompatible lox sites (loxP and lox2722) to generate a double floxedinverted open reading frame (D10) under the control of the elongationfactor 1a (EF-1α) promoter. All constructs are available from theDeisseroth Lab (www(dot)optogenetics(dot)org).

HEK293 cells were cultured in Dulbecco's minimal essential mediumsupplemented with 10% fetal bovine serum, 2 mM glutamine (Biochrome,Berlin, Germany), and 1% (w/w) penicillin/streptomycin. Cells wereseeded onto coverslips at a concentration of 0.175×106 cells/ml andsupplemented with 1 μM all-trans retinal. Transient transfection wasperformed with Fugene 6 (Roche, Mannheim, Germany) and recordings weredone 20-28 hours later. Photocurrents in transiently transfected HEK293cells were recorded by conventional whole-cell patch-clamp. The externalsolution contained [mM]: 140 NaCl, 2 CaCl₂, 2 MgCl₂, 2 KCl, 10 HEPES (pH7.2). The internal solution contained [mM]: 110 NaCl, 10 EGTA, 2 MgCl₂,1 CaCl₂, 5 KCl, 10 HEPES (pH was adjusted to 7.2 either using CsOH orHCl). Patch pipettes were pulled with micropipette puller model P-97(Sutter Instrument Co., Novato, Calif.) from microhaematocrit-tubes(Hecht-Assistant, Sondheim, Germany) with 1.5-2 MΩ resistance. HEK cellwhole-cell patch-clamping was performed with an EPC 7 (HEKA, ElektronikGmbH, Lambrecht, Germany) amplifier. Analog data was sampled at 20 kHz,digitized with Digidata1440 (Molecular Devices, Foster City, Calif.) anddisplayed using pClamp10.1 Software (Molecular Devices, Foster City,Calif.). For recording wavelength dependence, a light guide from aPolychrome V unit (TILL Photonics, Planegg, Germany) was mounted on theepiluminescence port of an Olympus IX70 microscope. For reflecting lightinto the objective a beam splitter (70% R/30% T) was used resulting in afinal photon density of ˜1×10₂₂ photons m⁻² s⁻¹ at 470 nm on thecoverslip. For recording the action spectra only 50% of the lightintensity was used. The polychrome V Unit was controlled with TillvisionSoftware (TILL Photonics, Planegg, Germany) synchronized with the pClampSoftware.

Results

Interestingly, the most robust improvement in chimeras was found wherehelix 1 and 2 were replaced with the homologs from ChR1 (FIG. 4). Twochimeric ChR1-VChR1 channels for membrane targeting and photocurrentsize were tested in cultured HEK293 cells. The first was joined in thesecond intracellular loop after Ala145 of ChR1, and the second wasjoined within helix three after Trp163 of ChR1 (FIG. 3). Whereas bothvariants were nearly equally well expressed in HEK293 cells (FIG. 4), incultured neurons the second variant expressed more robustly (FIG. 5) andshowed greatly enhanced peak photocurrents (888±128 pA, n=11 cells;p<0.0005) compared with VChR1-EYFP (FIG. 2). The action spectrum peakremained robustly redshifted relative to ChR2 (Table 1; FIG. 6), and theionic selectivity of the chimera was similar to that previously reportedfor ChR2 and VChR1 (FIG. 7) Adding the Kir2.1 trafficking sequence tothis hybrid trended to further increased photocurrents (1104±123 pA,n=12 cells; p<0.0005 compared with VChR1-EYFP, p=0.23 compared withC1V1-EYFP; FIG. 2; Tables 1-2). The resulting hybrid ChR1/VChR1 chimeracontains no ChR2 sequence at all, is remarkably derived from two opsingenes that do not express well alone, and is here referred to as C1V1(FIG. 1, FIG. 8).

Example 2 Optimization of Photocurrent Kinetics of C1V1

Fast deactivation properties²⁸ of this redshifted opsin would berequired for maximal temporal as well as spectral separation from otheropsins that are activated by wavelengths located towards the blue end ofthe visible spectrum. However, it was found that the photocurrentsdisplayed by C1V1-ts-EYFP exhibited >10-fold slower decay than ChR2, andeven slower decay than the original VChR1 (FIG. 9; To_(ff) 156±12 ms and132±12 ms for C1V1-ts-EYFP (n=4) and VChR1-EYFP (n=5), respectively;Table 1), potentially precluding the use of C1V1 for applicationsrequiring rapid firing rates. To correct the photocurrent kinetics ofC1V1, the chromophore region was searched using known structuralmodels^(22,28) (FIG. 8) for mutations with faster photocycle kinetics,reduced inactivation and reduced blue absorption. Next, glutamate-122was mutated to threonine, based on studies of the glutamate-rich motifin helix 2 showing that this mutation reduces inactivation.³

Materials and Methods

All point mutations in C1V1 vectors were generated in the plasmids bysite-directed mutagenesis (Agilent Technologies, Palo Alto, Calif.). Themembrane trafficking signal was derived from the K_(ir)2.1 channel.Mutations were confirmed by sequencing the coding sequence and splicesites.

Results

The ChETA-homologous mutation E162T²⁸ markedly accelerated thephotocycle almost 3-fold To_(ff) 58±4.7 ms, n=7 cells; FIG. 9, Table 1).Surprisingly, whereas analogous mutations in ChR2 or other microbialopsins have caused a red-shift^(28,29), in C1V1 this mutation shiftedthe action spectrum in the undesired direction, hypsochromic to 530 nm(FIG. 6; Table 1). C1V1-E122T inactivated only by 26% compared to 46%deactivation of ChR2 (FIG. 10, Table 1); in addition, the spectrum wasfurther red-shifted to 546 nm (FIG. 6, Table 1) and showed a strikingloss of the problematic blue shoulder of the C1V1 action spectrum.Finally, in the double mutant E122T/E162T, inactivation of the currentwas even lower than in the E122T mutant and the photocycle was stillfaster compared to E162T To_(ff) 34±4.9 ms, n=7 cells; FIG. 9, FIG. 11,Table 1), while preserving the redshift and absence of the blue shoulderof the action spectrum. Moreover, while the E122 mutant severely reducedphotocurrent amplitude (FIG. 12, the double mutant restored the veryhigh currents characteristic of the original C1V1-ts. Thus, multiplesurprising and useful properties of the individual mutations wereconserved in the double mutant, trafficking-enhanced C1V1 chimera.

TABLE 1 Spectral/kinetic properties of ChR2, VChR1 and C1V1 variants.Absorption Toff Kinetics Peak current (pA) Ratio maximum (nm) pH7.2 (ms)at −60 Mv* 405/560 Desensitation % ChR2 460 ± 6 10 ± 1  816 ± 18160%:8%  65 ± 8  (N = 5) (N = 5) (N = 5) (N = 7) (N = 5) VChR1 543 ± 7 85 ± 11 284 ± 54  9%:82% 53 ± 10 (N = 7) (N = 6) (N = 5) (N = 7)  (N =18) C1V1 539 ± 4 60 ± 6 1035 ± 158 28%:86% 46 ± 12  (N = 10) (N = 6) (N= 6)  (N = 10)  (N = 14) C1V1(E162T) 530 ± 4 23 ± 5 1183 ± 53  20%:71%41 ± 12 (N = 6) (N = 4) (N = 6) (N = 6) (N = 7) C1V1(E122T) 546 ± 5 55 ±8 572 ± 21 14%:94% 26 ± 6  (N = 4) (N = 5) (N = 5) (N = 4) (N 4)C1V1(E122T, 535 ± 5 12 ± 1 1072 ± 89  11%:72% 11 ± 9  E162T) (N = 7) (N= 5) (N = 9) (N = 7) (N = 9) Peak activation wavelength was recorded inHEK cells using 2 ms light pulses at the peak activation wavelength.Deactivation kinetics (τ_(off)) and peak photocurrents were recorded incultured hippocampal neurons using 2 ms light pulses at the maximalactivation wavelength. To identify the optimal variants forcombinatorial activation with ChR2, the percent response at 405 nm and560 nm was recorded in HEK cells. Desensitization of the photocurrentwas recorded using 300 ms light pulses, quantifying the decay of thepeak photocurrent (I_(max)) to the steady state.

TABLE 2 Summary of p-values from unpaired t-test comparisons for peakphotocurrent amplitude across all opsins shown in TABLE 1. Photocurrentswere recorded in cultured neurons using a 2 ms light pulse at 540 nm(VChR1 and C1V1 variants) or 470 nm (ChR2(H134R)). C1V1 C1V1 VchR1-VChR1-ts- C1V1- C1V1-ts- (E162T)- (E162T/E122T)- ChR2(H134R)- YFP YFPYFP YFP ts-Y ts-YFP YFP 1.0000 0.5770 0.0188 0.0029 6.5E−06 1.1E−050.0448 VChr1-YFP 1.0000 0.266 0.0039 1.1E−06 0.0015 0.0579 VChR1-ts-YFP1.0000 0.3372 0.0399 0.0788 0.8175 C1V1-YFP 1.0000 0.4099 0.8442 0.4222C1V1-ts-YFP 1.0000 0.3254 0.1490 C1V1(E162T)-ts-Y 1.0000 0.3001C1V1(E162T/E122T)- ts-YFP 1.0000 ChR2(H134R)-YFP

Thus, multiple useful properties of the individual mutations wereconserved together in the double mutant.

Example 3 Use of Novel C1V1 Chimeras in Prefrontal Cortex Neurons

To test these novel C1V1 opsin genes in neurons, lentiviral vectorsencoding C1V1-ts-EYFP and the point mutation combinations above weregenerated. These opsins were then expressed in cultured hippocampalneurons and recorded whole-cell photocurrents under identicalstimulation conditions (2 ms pulses, 542 nm light, 5.5 mW mm⁻²) todetermine whether the improvement in photocurrent amplitude resulteddirectly from the increased expression of C1V1 compared to VChR1.

Materials and Methods

Animals

Wild-type or transgenic Parvalbumin::Cre C57/BL6J male mice were grouphoused three to five to a cage and kept on a reverse 12 hour light/darkcycle with ad libitum food and water. Experimental protocols wereapproved by Stanford University IACUC and meet guidelines of theNational Institutes of Health guide for the Care and Use of LaboratoryAnimals.

Whole Cell Patch-Clamp Electrophysiology in Hippocampal and CorticalNeurons

Primary hippocampal cultures were isolated from PO Sprague-Dawley rats,plated on Matrigel (Invitrogen)-coated glass coverslips and treated withFUDR to inhibit glia overgrowth. Endotoxin-free plasmid DNA wastransfected in cultured neurons using a HEPES buffered Saline/CaPO4 mix.Electrophysiological recordings from individual neurons identified byfluorescent protein expression were obtained in Tyrode media ([mM] 150NaCl, 4 KCl, 2 MgCl2, 2 MgCl2, 10 D-glucose, 10 HEPES, pH 7.35 withNaOH) using a standard internal solution ([mM] 130 KGluconate, 10 KCl,10 HEPES, 10 EGTA, 2 MgCl2, pH 7.3 with KOH) in 3-5 MΩ glass pipettes.For cortical slice physiology, acute 300 gm coronal slices from 8-9 weekold wild-type C57BL/6J or PV::Cre mice previously injected with viruswere obtained in ice-cold sucrose cutting solution ([mM] 11 D-glucose,234 sucrose, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, 0.5 CaCl2, 26 NaHCO3)using a Vibratome (Leica). Slices were recovered in oxygenatedArtificial Cerebrospinal Fluid (ACSF; [mM] 124 NaCl, 3 KCl, 1.3 MgCl2,2.4 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 D-glucose) at 32° C. for onehour. Individual neuron patches were obtained after identifyingfluorescent protein expression from indicated prefrontal cortical layerunder constant ACSF perfusion. Filtered light from a broad-wavelengthxenon lamp source (Sutter Instruments DG-4) was coupled to thefluorescence port of the microscope (Leica DM-LFSA). Band pass filters(Semrock) had 20 nm bandwidth, and were adjusted with additional neutraldensity filters (ThorLabs) to equalize light power output across thespectrum.

Cultured cell images were acquired on the same microscope using a RetigaExi CCD camera (Qimaging, Inc.) at 100 ms exposure with 30 gain.Illumination power density was 12 mW mm⁻² at 500 nm with a standard EYFPfilter set. Quantification of fluorescence was performed with ImageJsoftware by marking a region containing the soma and proximal neuritesand calculating for each cell the total integrated pixel intensity inthat region, rather than average fluorescence, since photocurrents arelikely to be related to the total number of membrane-bound channelsrather than average channel expression per area.

Virus Preparation and Injection

Both Lentiviral- and AAV-mediated gene delivery were used forheterologous expression of opsins in mice. Indicated opsins were drivenby either Human calmodulin-dependent protein kinase II alpha (CaMKIIα)promoter to target cortical excitatory neurons or Elongation Factor 1a(EF-1a) in conjunction with a Cre-inducible cassette and followed by theWoodchuck hepatitis virus posttranscriptional regulatory element (WPRE).Cre-inducible recombinant AAV vector was produced by the University ofNorth Carolina Vector Core (Chapel Hill, N.C., USA) and used inconjunction with parvalbumin::Cre transgenic mice to target parvalbuminpositive nterneurons. Briefly, AAV constructs were subcloned into amodified version of the pAAV2-MCS, serotyped with AAV5 coat proteins andpackaged by the viral vector core at the University of North Carolina.The final viral concentration of AAV vectors as 1×10¹² genome copies(gc)/mL. Lentiviral constructs were generated as reported. Allconstructs are available from the Deisseroth Lab (www.optogenetics.org).Stereotactic viral injections were carried out under protocols approvedby Stanford University. Juvenile (4-6 weeks) mice kept under isofluraneanesthesia were arranged in a stereotactic frame (Kopf Instruments) andleveled using bregma and lambda skull landmarks. Craniotomies werepreformed so as to cause minimal damage to cortical tissue. Infralimbicprefrontal cortex (IL; from bregma: 1.8 mm anterior, 0.35 mm lateral,−2.85 mm ventral) was targeted using a 10 uL syringe and 35 g beveledneedle (Word Precision Instruments). Virus was infused at a rate of 0.1μL/min Subjects injected with virus for behavioral studies wereadditionally implanted with a chronic fiber optic coupling device tofacilitate light delivery either with or without an attached penetratingcerebral fiber for local delivery to target cortical region as noted(Doric Lenses, Canada). Penetrating fibers were stereotacticallyinserted to a depth of −2.5 mm from the same anterior and lateralcoordinates and affixed using adhesive luting cement (C&B MetaBond)prior to adhesive closure of the scalp (Vetbond, 3M) Animals wereadministered analgesic relief following recovery from surgery.

Results

Recordings from cultured hippocampal neurons expressing individualconstructs and an integrated fluorescence reading were obtained fromeach individual cell. In individual cells, fluorescence levels closelycorrelated with the measured photocurrent amplitudes across constructs(FIG. 13). It was therefore concluded that the potently increasedphotocurrent of C1V1 resulted chiefly from improved expression inneurons. Since the double mutant C1V1-E122T/E162T showed superiorperformance along all dimensions tested (photocurrent size, inactivationkinetics, and action spectrum), performance to ChR2(H134R) was alsodirectly compared by measuring integrated somatic YFP fluorescence andpeak photocurrents. Surprisingly, C1V1-E122T/E162T cells showed strongerphotocurrents than ChR2-H134R cells at equivalent fluorescence levels(FIG. 14), potentially suggestive of increased unitary conductance.

To examine whether C1V1-E122T/E162T would be suitable for opticallydriving spiking in pyramidal neurons, adeno-associated viral vectorsharboring the C1V1-E122T/E162T-ts-EYFP gene under the CaMKIIa promoter(AAV5-CaMKIIa-C1V1-E122T/E162T-ts-EYFP) were generated and injected thevirus into the prefrontal cortex of mice. Responses were recorded fromexpressing neurons in acute slices with 2 ms light pulse trains andcompared with responses to trains of current injection (10 ms, 200 pA)at identical frequencies. It was found that the frequency response ofneurons to 560 nm pulse trains was indistinguishable from the responseto current injections at the same frequencies (FIG. 15; n=6 cells in 2slices), suggesting that intrinsic properties of the cell and not C1V1kinetics limit spiking performance at high rates. Similar performanceproperties were seen across a range of green, yellow, and amberillumination conditions, with strong performance at the moderate lightintensities (<10 mW/mm²) suitable for in vivo applications in mammals(FIG. 16). Indeed, responses at 540 nm and 590 nm were similarlyeffective at evoking precisely timed action potentials, with lowerfidelity at lower light powers as expected (FIG. 16).

With the prominently red-shifted action spectrum, the possibility thatC1V1 might even be used to drive spiking with red light, not previouslyreported with any opsin and potentially important for allowing improvedspectral separation as well as control of neurons in deeper tissue, wasconsidered. Whether any C1V1 variants could be used to drive spikesusing far-red light was therefore examined Although the kinetics ofC1V1-E122T were slower than those of C1V1-E122T/E162T, its actionspectrum was the most red-shifted of all variants (FIG. 6), and indeedit was found that cells expressing C1V1-E122T responded more strongly tored light (630 nm) than cells expressing the double mutant (FIG. 17,top). Although on-kinetics of the E122T mutant at 630 nm were slowerthan at 545 nm (FIG. 18), photocurrents were recruited using longerpulses of 630 nm light at moderate intensity (FIG. 19) that sufficed toelicit defined spike trains (FIG. 20; FIG. 17, bottom).

Example 4 Use of Novel C1V1 Chimeras in Living Brain Slices from thePrefrontal Cortex Neurons of Mice

The study sought to determine whether inhibitory and excitatory neuronsresiding within the same microcircuit could be targeted with theintroduction of C1V1 variants. Independent activation of two neuronalpopulations within living brain slices was explored; in this caseCaMKIIα-C1V1-E122T/E162Tts eYFP and EF1a-DIO-ChR2-H134R-EYFP wereexpressed in mPFC of PV::Cre mice.

Materials and Methods

Acute 300 μm coronal slices isolated from 8-9 week old wild-typeC57BL/6J or PV::Cre mice previously injected with virus were obtained inice-cold sucrose cutting solution ([nM] 11 D-glucose, 234 sucrose, 2.5KCl, 1.25 NaH₂PO₄, 10 MgSO₄, 0.5 CaCl₂, 26 NaHCO₃) using a Vibratome(Leica). Slices were recovered in oxygenated Artificial CerebrospinalFluid (ACSF; [mM] 124 NaCl, 3 KCl, 1.3 MgCl₂, 2.4 CaCl₂, 1.25 NaH₂PO₄,26 NaHCO₃, 10 D-glucose) at 32° C. for one hour. Individual neuronpatches were obtained after identifying fluorescent protein expressionfrom indicated prefrontal cortical layer under constant ACSF perfusion.Filtered light from a broad-wavelength xenon lamp source (SutterInstruments DG-4) was coupled to the fluorescence port of the microscope(Leica DM-LFSA). Slice physiology data were imported into Matlab andanalyzed using custom-written software. Power spectra were calculatedusing the wavelet method as described by Sohal et al.⁵⁵ Briefly, foreach frequency f, the recorded traces were first filtered with abandpass filter between f±5 Hz. The filtered traces were then convolvedwith the wavelet function:W(f,t)=s(t)*9(f,t)G(f,t)=e ^((−t) ² ^()(2σ) ² ^(e) ^(−πift) ⁾ ⁻¹where * denotes convolution, σ=5/(6f). The squared amplitude of W(f,t)over a 500 msec window was then used to measure the power at variousfrequencies. All power spectra from slice recordings were normalized to1/f.

Cultured cell images were acquired on the same microscope using a RetigaExi CCD camera (Qimaging inc.) at 100 ms exposure with the 30 gain.Illumination power density was 12 mW mm⁻² at 500 nm with a standard EYFPfilter set. Quantification of fluorescence was done with ImageJ softwareby marking a region containing the soma and proximal neuritis andcalculating for each cell the total integrated pixel intensity in thatregion, rather than average fluorescence, since photocurrents are likelyto be related to the total number of membrane-bound channels rather thanaverage channel expression per area.

Using current clamp, a single pyramidal cell was stimulated with a trainof simulated EPSC waveforms. Individual sEPSC events had peak currentmagnitudes of 200 pA and decayed with a time constant of 2 ms. Eachexperiment was divided into 10 sweeps, each 10 seconds long andseparated by 5 seconds to minimize rundown. Each sweep was divided into500 ms segments. The total number of sEPSCs in each 500 ms segment wasrandomly chosen from a uniform distribution between 0 and 250. Then, thetimes of the sEPSCs within the 500 ms segment were randomly selectedfrom a uniform distribution extending across the entire segment,simulating excitatory input from a population of unsynchronized neurons.Empirically, these stimulation parameters reliably drove pyramidalneurons at firing rates from 0-30 Hz. In conditions marked as baseline,a 10 sec pulse of 590 nm light was delivered to completely inactivatethe opsin before running the sEPSC protocol. In conditions where theopsin was activated, a 1 sec pulse of 470 nm light preceded the sEPSCprotocol.

To understand the net effect of altered E/I balance on informationprocessing, the mutual information between each neuron's input sEPSCrate and output spike rate, which captures relevant changes in the shapeof the I-0 curve and in the response variability was computed. First,the joint distribution of sEPSC rate and spike rate by binning in time,sEPSC rate, and spike rate were estimated and the building of a jointhistogram. Time bins were 125 ms wide, and sEPSC rate was divided into10 equally spaced bins from 0 to 500 Hz, although the mutual informationresults were consistent across a wide range of binning parameters. Spikerate was binned using the smallest meaningful bin width given the timebin width (e.g. 8 Hz bin width for 125 ms time bins). From this jointhistogram, compute mutual information, as previously described wascomputed equaling the difference between response entropy and noiseentropy.

Response entropy quantifies the total amount of uncertainty in theoutput spike rate of the neuron. Noise entropy quantifies theuncertainty that remains in the output spike rate given the input rate.Note that the maximum information that neural responses can transmitabout the input stimulus is the entropy of the stimulus set. For 10equally spaced input sEPSC rate bins and a uniform distribution of inputrate over these bins, the entropy of the input rate is log₂(10)=3.322bits.

Mutual information calculated from undersampled probabilitydistributions can be biased upwards. Consequently, all reported valuesof mutual information, response entropy and noise entropy were correctedfor bias due to undersampling. This correction is done by computingvalues from smaller fractions (from one-half to one-eighth) of the fulldata and extrapolating to the limit of infinite data. Using 125 ms timewindows, the correction factors were always less than 0.07 bits.

Vectors were created and injections were performed as above.

Results

Using this array of multiply engineered opsin genes, the possibilitiesfor combinatorial control of cells and projections within intactmammalian systems was explored. First, it was asked whether excitatoryand inhibitory neurons residing within the same microcircuit could beseparably targeted by the respective introduction of C1V1 variants andconventional ChRs into these two cell populations. It was found thatcultured hippocampal neurons expressing C1V1-E122T/E162T spiked inresponse to 2 ms green light pulses (560 nm) but not violet lightpulses. In contrast, cells expressing ChR2-H134R spiked in response to 2ms 405 nm light pulses, but not in response to 2 ms 561 nm light pulses(FIG. 21). This principle was therefore tested within living brainslices; in this case AAV5-CaMKIIa::C1V1-E122T/E 162T-ts-mCherry alongwith AAV5-EFla-DIO::ChR2-H134R-EYFP in was expressed in mPFC of PV::Cremice (FIG. 22). In pyramidal neurons not expressing any opsin, 405 nmlight pulses triggered robust and fast inhibitory postsynaptic currentsdue to direct activation of PV cells (FIG. 23), while 561 nm lightpulses triggered both short-latency EPSCs (FIG. 24) and the expectedlong-latency polysynaptic IPSCs arising from C1V1-expressing pyramidalcell drive of local inhibitory neurons (FIG. 23).

Excitation of these independent cellular elements in vivo with optroderecordings was then explored (FIG. 25, left). To examine the inhibitoryeffect of PV cell activity on pyramidal neuron spiking, an experimentalprotocol in which 5 Hz violet light pulses (to activate ChR2 in PVcells) preceded 5 Hz green light pulses (to activate C1V1 in excitatorypyramidal neurons) with varying inter-pulse intervals was designed. Whenviolet and green light pulses were separated by 100 ms (FIG. 25, toptrace), responses to green light pulses were not affected by the violetpulses. However, as delays between violet and green pulses were reduced,green light-induced events became more readily inhibited and werecompletely abolished when light pulses were presented with sufficientsynchrony (FIG. 25, bottom trace; summary data in FIG. 26). These datademonstrate combinatorial optogenetic activation within an intact mammal(driving one population alone or in precise temporal combination withanother), capitalizing on the speed of the opsins and the properties ofthe delivered light pulses.

Example 5 Effect of Independent Activation of Corticothalamic (CT) andThalamocortical (TC) Glutamatergic Axons Impinging Upon Neurons of theReticular Thalamic Nucleus

To validate the combinatorial control property for axonal projectionsinstead of direct cellular somata stimulation, the effect of independentactivation of corticothalamic (CT) and thalamocortical (TC)glutamatergic axons impinging upon neurons of the reticular thalamicnucleus (nRT) (FIG. 28) was examined in thalamic slices.

Materials and Methods

C57BL/6J wild-type (postnatal days 90-120) were anesthetized withpentobarbital (100 mg/kg, i.p.) and decapitated. The thalamic slicepreparation and whole-cell patch-clamp recordings were performed.Recordings were obtained from nRT (reticular thalamic) and TC (relaythalamocortical) neurons visually identified using differential contrastoptics with a Zeiss (Oberkochen, Germany), Axioskop microscope, and aninfrared video camera. For EPSCs and current-clamp recordings, theinternal solution contained (in mM): 120 Kgluconate, 11 KCl, 1 MgCl₂, 1CaCl₂, 10 Hepes, 1 EGTA. pH was adjusted to 7.4 with KOH (290 mOsm).E_(Cl) ⁻ was estimated ˜−60 mV based on the Nernst equation. Potentialswere corrected for −15 mV liquid junction potential. For voltage-clampexperiments neurons were clamped at −80 mV and EPSCs werepharmacologically isolated by bath application of the GABAA receptorantagonist picrotoxin (50 μM, Tocris). In all recording conditions,access resistance was monitored and cells were included for analysisonly if the access resistance was <18 MΩ and the change of resistancewas <25% over the course of the experiment.

600 nL rAAV5/CamKIIα-hChR2(H134R)-EYFP or 900 nLrAAV5-CaMKIIαC1V1(E122T/E162T)-TS-mCherry virus was injectedstereotaxically into ventrobasal thalamus (VB) or barrel cortex,respectively, of C57BL/6J wild-type mice in vivo, between post-nataldays 30-35. Intra-cortical and intra-thalamic (VB) injections wereperformed in the same mice (n=6). Intra-cortical injections werepreformed (from bregma) 1.3 mm posterior, 3 mm lateral, 1.15 mm belowthe cortical surface. Intra-thalamic injections were 1.7 mm posterior,1.5 mm lateral, 3.5 mm below the cortical surface. Mice were sacrificed˜2-3 months following injections and horizontal brain thalamic sliceswere made for optical stimulation and in vitro recordings as describedabove. VB thalamus was removed to avoid disynaptic activation of nRTneurons via the CT-TC-nRT pathway. Cutting VB thalamus from slicesremoved all photosensitive cell bodies from the preparation, enableddirect examination of CTnRT and TC-nRT projections, and did not affectthe electrical membrane properties of nRT neurons (not shown). Opticalactivation of ChR2-expressing TC and C1V1-expressing CT axons wereperformed with 405 nm and 560 nm laser stimuli, respectively (5 msduration light pulses, 2-3 mW) (OEM Laser Systems, MI) delivered withoptic fiber (BFL 37-300, ThorLabs) upstream along the CT and TC pathwaysprojecting to nRT. Minimal stimulation intensity was used, defined asthe light power that resulted in 50 to 70% failures (30-50% successes),fixed response kinetics and low response amplitude variability.Consequent minimal evoked EPSCs presumably resulted from selectiveoptical activation of single CT or TC axons presynaptic to the recordedcell. The stimulation light power was slightly increased (˜5% aboveminimal stimulation) until the number of failures became 0. CT and TCinputs were (simultaneously) stimulated and minimal evoked EPSCs andEPSPs (each individually subthreshold for action potential firing) wererecorded in nRT cells.

Statistical significance was calculated using paired or unpairedtwo-tailed t-tests, as applicable. Data were analyzed using MatlabStatistics toolbox or Microsoft Excel

Results

Minimal stimulation of TC axons evoked large and fast excitatorypost-synaptic currents (EPSCs) in nRT neurons, whereas minimalstimulation of CT axons evoked small and slow EPSCs in nRT neurons (FIG.30), both typical for these pathways.

Next the synaptic integration of CT and TC inputs under variable delayconditions between these two inputs was examined. Subthreshold EPSPsfrom each pathway became suprathreshold for action potential firing onlywhen coincident within 5 ms (FIG. 29, FIG. 31). The temporal precisionof C1V1 and ChR2 activation allowed a reliable control of the delaybetween the TC and CT inputs and thus allowed determination of a narrowwindow (−5 ms) of effective synaptic integration in nRT cells, notpreviously observable with existing electrical, pharmacological, oroptogenetic techniques due to the reciprocal connectivity of cortex andthalamus as well as the close approximation of CT and TC axons. Theseresults demonstrate for the first time, in the same intact preparation,independent activation of distinct axonal projections to examine theircombinatorial effects on the same target cell.

Example 6 Use of C1V1 and SSFO to Achieve Spectrotemporal Separation ofNeural Activation within the Same Circuit

In both of the above two preparations, visible-spectrum violet (405 nm)and green (560 nm) lasers were used to achieve separable activation ofthe two opsins. While 405 nm lasers deliver safe non-UV light, for manyapplications it may be preferable to use 470 nm laser light for theblue-responsive opsin, since 470 nm light will penetrate tissue moredeeply, scatter less, and be more easily and economically delivered fromcommon blue light sources. While this may seem impossible since 470 nmlight will partially activate C1V1 (FIG. 7) as well as ChR2,combinatorial control could be achievable even with 470 nm light,capitalizing on both the temporal properties of SSFO and the redshiftednature of C1V1 to achieve “spectrotemporal separation” within intactmammalian tissue. To test this possibility, it was decided to directlycompare, within the same preparation, the effects on rhythmic activityof stably potentiating either excitatory or inhibitory cells (FIG. 33)

Materials and Methods

ChR2-D156A and SSFO were generated by inserting point mutations into thepLenti-CaMKIIα

ChR2-EYFP-WPRE vector using site-directed mutagenesis (Quikchange II XL;Stratagene). Viral gene delivery, coronal brain sectioning, and patchclamp recording were performed as above. Double virus injections toexpress CaMKIIa::C1V1 and DIO-SSFO in the mPFC of PV::Cre mice wereperformed.

While handling cells or tissues expressing SSFO, care was taken tominimize light exposure to prevent activation by ambient light. Beforeeach experiment, a 20 s pulse of 590 nm light was applied to convert allof the SSFO channels to the dark state and prevent rundown ofphotocurrents. For acquisition of SSFO activation and deactivationspectra, recordings from cultured neurons were made in voltage clampmode. For recording activation spectra, a 1 s pulse of varyingwavelength was applied, followed by a 10 s 590 nm pulse. Deactivationspectra were acquired by first applying a 1 s 470 nm pulse to activateSSFO, followed by a 10 s pulse of varying wavelength. Net activation ordeactivation was calculated by dividing the photocurrent change afterthe first or second pulse, respectively, by the maximum photocurrentchange induced by the peak wavelength for that cell. Negative values indeactivation spectra resulted from traces in which, for example, a 10 s470 nm pulse led to a slight increase in photocurrent rather thandeactivate the channels. This could be the result of the relatively wide(20 nm) band-pass filter width used for these recordings with the SutterDG-4. Intermediate wavelengths (between 470 nm and 520 nm) are expectedto have a mixed effect on the channel population for the same reasons.

Photon flux calculations for SSFO integration properties were conductedby calculating the photon flux through the microscope objective at eachlight power, and then dividing to reach the photon flux across the cellsurface, based on the diameter of the recorded cells and approximatingcell shape as a spheroid.

Results

SSFO is a novel multiply-engineered channelrhodopsin with a decayconstant of 29 minutes that can be effectively deactivated at the samewavelengths that activate C1V1 and permits bistable excitation ofneurons over many minutes with enhanced light sensitivity. Informationregarding SSFOs can be found in International Patent ApplicationPublication No: WO 2010/056970 and United States Patent Application No.61/410,704 and 61/410,711, the contents of which are hereby incorporatedby reference herein in their entireties. Double virus injections toexpress CaMKIIa::C1V1 and DIO::SSFO in acute slices from the mPFC ofPV::Cre mice were performed. Under these conditions, excitatorypyramidal cells should respond to redshifted light, and inhibitory PVcells to blue light. Indeed, in response to a 1 s 470 nm light pulse toactivate SSFO in the PV cells, the rate of ongoing IPSCs was stablyincreased from 8.5±1.2 Hz at baseline (period 3, FIG. 34) to 16.8±2.1 Hzafter the blue light pulse (period 2; n=4 recordings, p<0.005, pairedt-test; FIG. 35), showing persistent activation of the SSFO-expressinginhibitory cells. Even though 470 nm light will also transientlyactivate C1V1, this activation can only occur during the light pulseitself due to the very fast deactivation of C1V1 after light-off; theprolonged post-light period is characterized by SSFO activity only (FIG.34), illustrating temporal separation of optogenetic control modes.Interestingly, during this prolonged period of elevated PV neuronactivity, no significantly elevated peak in the IPSC power spectrum waselicited, suggesting that direct activation of PV neurons alone in thisreduced preparation is insufficient to elicit gamma synchrony in thenetwork. However, in marked contrast, during the 470 nm light pulseitself when the same level of PV neuron activation but also partialactivation of C1V1-expressing pyramidal cells is also expected, apronounced gamma peak was consistently observed (peak frequency 39.2±3.5Hz; n=4 recordings;) that extended into the high-gamma range (>60 Hz).

Moreover, in the same experiments (indeed, later in the same recordedsweeps), direct activation in this case of C1V1-pyramidal cells alonewith 590 nm light (which simultaneously activates C1V1 in PY cells anddeactivates the SSFO in PV cells) led to robust gamma synchrony, with alower frequency peak (26.6±1 Hz, n=6 recordings). Demonstrating that anyresidual PV neuron activity linked to the prior history of SSFOactivation in PV cells was not necessary for this effect,otherwise-identical sweeps with only a history of C1V1 activation in thepyramidal cells and no prior history of elevated IPSC rate elicited thesame result). These results illustrate the integrated principle ofspectrotemporal combinatorial control, and also suggest that elevatingactivity in pyramidal neurons can give rise through network propertiesto gamma oscillations³¹. Interestingly, during the 470 nm light pulse,when activation of both PV and pyramidal cells was expected, gammasynchrony was consistently observed at higher frequencies than when onlyexcitatory neurons were activated, supporting and extending informationon the coordinated importance of both PV and pyramidal cells ineliciting gamma oscillations.³¹⁻³³

CONCLUSION

In the course of this work, a family of new tools was generated that arereferred to as C1V1 variants. C1V1 is a red-shifted opsin geneassembled, remarkably, from pieces of other opsin genes that do notexpress well alone in neurons, but which were identified in earliergenomic searches (VChR1 and ChR1). C1V1 contains no ChR2 sequence atall, yet its multiply-engineered variants reported here now representthe most potent, most redshifted, and most stable channelrhodopsinsknown. Mutagenesis in key amino acid positions throughout the retinalbinding pocket led to the generation of (1) C1V1(E162T), ahigh-expressing redshifted opsin gene generated as a fast homolog of theChETA mutation; (2) C1V1(E122T) which displays the reddest actionspectrum shoulder and can even be used to fire action potentials withred light (3) C1V1(E122T/E162T)—a combination mutant with the lowestdesensitization, fastest deactivation, least violet-light activation forminimal cross-activation with ChR2, and strong expression. Indeed, C1V1variants may be selected for different applications based onconsiderations of current size, deactivation kinetics, and actionspectrum (Table 1)—for example, in two-photon work, since 2P activationof ChR2 has been difficult due to current size and rapid kinetics ofchannel closure, C1V1(E162T) is likely to be of interest. The C1V1variants enabled direct testing of the hypothesis that increasing levelsof elevated cellular E/I balance would give rise to increasingintensities of gamma rhythmicity, a phenomenon previously linked to bothschizophrenia and autism. Of course, the different tools are alsosynergistic; using C1V1 variants together with ChR2 permitted reliableand separate driving of spiking in the two distinct neuronal populationsaddressed in this study—the excitatory pyramidal neurons and thefast-spiking, parvalbumin-expressing inhibitory interneurons, andconfirm that steady elevated cellular E/I balance was effective atgenerating gamma-band circuit activity, capitalizing on both kinetic andspectral differences in the optogenetic tools. This type ofcombinatorial activation can be extended beyond multiple cell types tomultiple neural pathway types—for example, the separable activation ofspiking, within a single brain region, in two converging axonal afferentpathways arising from distinct locations—a long-sought goal of systemsneuroscience.

The examples, which are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway, also describe and detail aspects and embodiments of the inventiondiscussed above. The foregoing examples and detailed description areoffered by way of illustration and not by way of limitation. Allpublications, patent applications, and patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or patent were specifically andindividually indicated to be incorporated by reference. Variousembodiments described above, and discussed in the attached Appendicesmay be implemented together and/or in other manners. One or more of theaspects in the present disclosure and in the Appendices can also beimplemented in a more separated or integrated manner, as should beapparent and is useful in accordance with particular targetapplications. In particular, all publications and appendices citedherein are expressly incorporated herein by reference for the purpose ofdescribing and disclosing compositions and methodologies which might beused in connection with the invention. Although the foregoing inventionhas been described in some detail by way of illustration and example forpurposes of clarity of understanding, it will be readily apparent tothose of ordinary skill in the art in light of the teachings of thisinvention that certain changes and modifications may be made theretowithout departing from the spirit or scope of the appended claims.

REFERENCES

-   Deisseroth, K. Optogenetics. Nat Methods 8, 26-29 (2011). Boyden, E.    S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K.    Millisecond-timescale, genetically targeted optical control of    neural activity. Nat Neurosci 8, 1263-1268 (2005).-   Nagel, G. et al. Light activation of channelrhodopsin-2 in excitable    cells of Caenorhabditis elegans triggers rapid behavioral responses.    Curr Biol 15, 2279-2284 (2005).-   Li, X. et al. Fast noninvasive activation and inhibition of neural    and network activity by vertebrate rhodopsin and green algae    channelrhodopsin. Proc Natl Acad Sci USA 102, 17816-17821 (2005).-   Bi, A. et al. Ectopic expression of a microbial-type rhodopsin    restores visual responses in mice with photoreceptor degeneration.    Neuron 50, 23-33 (2006).-   Schroll, C. et al. Light-induced activation of distinct modulatory    neurons triggers appetitive or aversive learning in Drosophila    larvae. Curr Biol 16, 1741-1747 (2006).-   Zhang, F. et al. Multimodal fast optical interrogation of neural    circuitry. Nature 446, 633-639 (2007).-   Douglass, A. D., Kraves, S., Deisseroth, K., Schier, A. F. &    Engert, F. Escape behavior elicited by single,    channelrhodopsin-2-evoked spikes in zebrafish somatosensory neurons.    Curr Biol 18, 1133-1137 (2008).-   Hagglund, M., Borgius, L, Dougherty, K. J. & Kiehn, 0. Activation of    groups of excitatory neurons in the mammalian spinal cord or    hindbrain evokes locomotion. Nature neuroscience 13, 246-252,    doi:10.1038/nn.2482 (2010).-   Huber, D. et al. Sparse optical microstimulation in barrel cortex    drives learned behaviour in freely moving mice. Nature 451, 61-64    (2008).-   Hira, R. et al. Transcranial optogenetic stimulation for functional    mapping of the motor cortex. J Neurosci Methods 179, 258-263 (2009).-   Higley, M. J. & Sabatini, B. L. Competitive regulation of synaptic    Ca2+ influx by D2 dopamine and A2A adenosine receptors. Nature    neuroscience 13, 958-966, doi:10.1038/nn.2592 (2010).-   Petreanu, L., Huber, D., Sobczyk, A. & Svoboda, K.    Channelrhodopsin-2-assisted circuit mapping of long-range callosal    projections. Nat Neurosci 10, 663-668 (2007).-   Ishizuka, T., Kakuda, M., Araki, R. & Yawo, H. Kinetic evaluation of    photosensitivity in genetically engineered neurons expressing green    algae light-gated channels. Neurosci Res 54, 85-94 (2006).-   Nagel, G. et al. Channelrhodopsin-2, a directly light-gated    cation-selective membrane channel. Proc Natl Acad Sci USA 100,    13940-13945 (2003).-   Rickgauer, J. P. & Tank, D. W. Two-photon excitation of    channelrhodopsin-2 at saturation. Proc Natl Arad Sci USA 106,    15025-15030 (2009).-   Yonehara, K. et al. Spatially asymmetric reorganization of    inhibition establishes a motion-sensitive circuit. Nature 469,    407-410, doi:10.1038/nature09711 (2011).-   Yaroslaysky, A. N. et al. Optical properties of selected native and    coagulated human brain tissues in vitro in the visible and near    infrared spectral range. Phys Med Biol 47, 2059-2073 (2002).-   Wang, H. et al. Molecular determinants differentiating photocurrent    properties of two channelrhodopsins from chlamydomonas. J Biol Chem    284, 5685-5596 (2009).-   Wen, L. et al. Opto-current-clamp actuation of cortical neurons    using a strategically designed channelrhodopsin. PLoS One 5, e12893    (2010).-   Zhang, F. et al. Red-shifted optogenetic excitation: a tool for fast    neural control derived from Volvox carteri. Nat Neurosci 11, 631-633    (2008).-   Berndt, A., Yizhar, O., Gunaydin, L. A., Hegemann, P. &    Deisseroth, K. Bi-stable neural state switches. Nat Neurosci 12,    229-234 (2009).-   Barnann, C., Gueta, R., Kleinlogel, S., Nagel, G. & Bamberg, E.    Structural guidance of the photocycle of channelrhodopsin-2 by an    interhelical hydrogen bond. Biochemistry 49, 267-278 (2010).-   Schoenenberger, P., Gerosa, D. & Oertner, T. G. Temporal control of    immediate early gene induction by light. PLoS One 4, e8185 (2009).-   Stehfest, K., Ritter, E., Berndt, A., Bartl, F. & Hegernann, P. The    branched photocycle of the slow-cycling channelrhodopsin-2 mutant    C128T. J Mol Biol 398, 690-702 (2010).-   Sohal, V. S., Zhang, F., Yizhar, 0. & Deisseroth, K. Parvalbumin    neurons and gamma rhythms enhance cortical circuit performance.    Nature 459, 698-702 (2009).-   Lin, J. Y., Lin, M. Z., Steinbach, P. & Tsien, R. Y.    Characterization of engineered channelrhodopsin variants with    improved properties and kinetics. Biophys J 96, 1803-1814 (2009).-   Gunaydin, L. A. et al. Ultrafast optogenetic control. Nat Neurosci    13, 387-392 (2010).-   Tittor, J., Schweiger, U., Oesterhelt, D. & Bamberg, E. Inversion of    proton translocation in bacteriorhodopsin mutants D85N, D85T, and    D85,96N. Biophys J 67, 1682-1690 (1994).-   Sugiyama, Y. et al. Photocurrent attenuation by a single    polar-to-nonpolar point mutation of channelrhodopsin-2. Photochem    Photobiol Sci 8, 328-336 (2009).-   Adesnik, H. & Scanziani, M. Lateral competition for cortical space    by layer-specific horizontal circuits. Nature 464, 1155-1160 (2010).-   Colgin, L. L. et al. Frequency of gamma oscillations routes flow of    information in the hippocampus. Nature 462, 353-357 (2009).-   Cardin, J. A. et al. Driving fast-spiking cells induces gamma rhythm    and controls sensory responses. Nature 459, 663-667 (2009).-   Bamann, C., Kirsch, T., Nagel, G. & Bamberg, E. Spectral    characteristics of the photocycle of channelrhodopsin-2 and its    implication for channel function. J Mol Biol 375, 686-694 (2008).

SEQUENCES (Humanized C1V1 amino acid sequence) SEQ ID NO: 1MSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERMLFQTSYTLENNGSVICIPNNGQCFLAWLKSNGTNAEKLAEKLAANILQWITFALSALCLMFYGYQTWKSTCGWEEIYVATIEMIKFIIEYFHEFDEPAVIYSSNGNKTVWLRYAEWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGCIVWGATSAMCTGWTKILFFLISLSYGMYTYFHAAKVYIEAFHTVPKGICRELVRVMAWTFFVAWGMFPVLFLLGTEGFGHISPYGSAIGHSILDLIAKNMWGVLGNYLRVKIHEHILLYGDIRKKQKITIAGQEMEVETLVAEEED (Humanized C1V1 nucleotide sequence) SEQ ID NO: 2atgtcgcgacgcccgtggctccttgctctcgcattggcggtggcgcttgcagcgggatcggcaggagcgtcaaccggaagcgatgcgaccgtccccgtggctacgcaagacggaccagattacgtgttccacagagcccacgagcggatgttgtttcagacatcatacacacttgaaaacaatggtagcgtcatttgcatccctaacaatgggcagtgtttttgcctggcctggttgaaatcgaacggtacgaacgccgagaagctggcggcgaacattctgcagtggatcacattcgcactctcggcgctctgccttatgttctatggctaccagacttggaaatccacgtgtggttgggaagagatctacgtagcaaccattgaaatgatcaagtttatcattgagtatttccatgagtttgacgaaccggccgtaatctactcatcgaatgggaataagacagtctggttgaggtatgcggagtggctcctcacctgcccggtccttctgatccatctgagcaacctcacggcctgaaggacgattatagcaaaaggactatgggcctgttggtttctgatgtgggatgcatcgtgtggggcgaaccagcgccatgtgtacggggtggacgaagatcctgttcttcctcatctcattgagctatggtatgtatacctattttcatgctgctaaagtttatatcgaagcattccacacagttccaaaagggatttgtcgagaactggtccgagtgatggcctggacattctttgtggcttggggaatgtttccagtcctgtttctgctgggcacggaaggattcggtcatatcagcccttatggatctgccattgggcactccatcctcgacctgattgcaaagaacatgtggggtgtgctggggaattacctgcgcgtcaaaatccacgagcacatcctgttgtatggcgacatcagaaagaagcagaaaattacgatcgccggccaagagatggaggttgagacactggtggctgaagaggaggactaa(Humanized C1V1 E122T amino acid sequence) SEQ ID NO: 3MSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERMLFQTSYTLENNGSVICIPNNGQCFCLAWLKSNGTNAEKLAANILQWITFALSALCLMFYGYQTWKSTCGWETIYVATIEMIKFIIEYFHEFDEPAVIYSSNGNKTVWLRYAEWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGCIVWGATSAMCTGWTKILFFLISLSYGMYTYFHAAKVYIEAFHTVPKGICRELVRVMAWTFFVAWGMFPVLFLLGTEGFGHISPYGSAIGHSILDLIAKNMWGVLGNYLRVKIHEHILLYGDIRKKQKITIAGQEMEVETLVAEEED(Humanized C1V1 E122T nucleotide sequence) SEQ ID NO: 4atgtcgcgacgcccgtggctccttgctctcgcattggcggtggcgcttgcagcgggatcggcaggagcgtcaaccggaagcgatgcgaccgtccccgtggctacgcaagacggaccagattacgtgttccacagagcccacgagcggatgttgtttcagacatcatacacacttgaaaacaatggtagcgtcatttgcatccctaacaatgggcagtgtttttgcctggcctggttgaaatcgaacggtacgaacgccgagaagctggcggcgaacattctgcagtggatcacattcgcactctcggcgcctgccttatgttctatggctaccagacttggaaatccacgtgtggttgggaaaccatctacgtagcaaccattgaaatgatcaagtttatcattgagtatttccatgagtttgacgaaccggccgtaatctactcatcgaatgggaataagacagtctggttgaggtatgcggagtggctcctcacctgcccggtccttctgatccatctgagcaacctcacaggcctgaaggacgattatagcaaaaggactatgggcctgttggtttctgatgtgggatgcatcgtgtggggcgcaaccagcgccatgtgtacggggtggacgaagatcctgttcttcctcatctcattgagctatggtatgtatacctattttcatgctgctaaagtttatatcgaagcattccacacagttccaaaagggatttgtcgagaactggtccgagtgatggcctggacattctttgtggcttggggaatgtttccagtcctgtttctgctgggcacggaaggattcggtcatatcagcccttatggatctgccattgggcactccatcctcgacctgattgcaaagaacatgtggggtgtgctggggaattacctgcgctcaaaatccacgagcacatcctgttgtatggcgacatcagaaagaagcagaaaattacgatcgccggccaagagatggaggttgagacactggtggctgaagaggaggactaa(Humanized C1V1 E162T amino acid sequence) SEQ ID NO: 5MSRRPWLLALALAVALAAGSAGASTGSGATVPVATQDGPDYVFHRAHERMLFQTSYTLENNGSVICIPNNGQCFCLAWLKSNGTNAEKLAANILQWITFALSALCLMFGYQTWKSTCGWEEIYVATIEMIKFIIEYFHEFDEPAVIYSSNGNKTVWLRYATWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGCIVWGATSAMCTGWTKILFFLISLSYGMYTYFHAAKVYIEAFHTVPKGICRELVRVMAWTFFVAWGMFPVLFLLGTEGFGHISPYGSAIGHSILDLIAKNMWGVLGNYLRVKIHEHILLYGDIRKKQKITIAGQEMEVETLVAEEED (Humanized C1V1 E162T nucleotide sequence) SEQ ID NO: 6atgtcgcgacgcccgtggctccttgctctcgcattggcggtggcgcttgcagcgggatcggcaggagcgtcaaccggaagcgatgcgaccgtccccgtggctacgcaagacggaccagattacgtgttccacagagcccacgagcggatgttgtttcagacatcatacacacttgaaaacaatggtagcgtcatttgcatccctaacaatgggcagtgtttttgcctggcctggttgaaatcgaacggtacgaacgccgagaagctggcggcgaacattctgcagtggatcacattcgcactctcggcgctctgccttatgttctatggctaccagacttggaaatccacgtgtggttgggaagagatctacgtagcaaccattgaaatgatcaagtttatcattgagtatttccatgagtttgacgaaccggccgtaatctactcatcgaatgggaataagacagtctggttgaggtatgcgacgtggctcctcacctgcccggtccttctgatccatctgagcaacctcacaggcctgaaggacgattatagcaaaaggactatgggcctgttggtttctgatgtgggatgcatcgtgtggggcgcaaccagcgccatgtgtacggggtggacgaagatcctgttcttcctcatctcattgagctatggtatgtatacctattttcatgctgctaaagtttatatcgaagcattccacacagttccaaaagggatttgtcgagaactggtccgagtgatggcctggacattctttgtggcttggggaatgtttccagtcctgtttctgctgggcacggaaggattcggtcatatcagcccttatggatctgccattgggcactccatcctcgacctgattgcaaagaacatgtggggtgtgctggggaattacctgcgcgtcaaaatccacgagcacatcctgttgtatggcgacatcagaaagaagcagaaaattacgatcgccggccaagagatggaggttgagacactggtggctgaagaggaggactaa(Humanized C1V1 E122T/E162T amino acid sequence) SEQ ID NO: 7MSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPYVFHRAHERMLFQTSYTLENNGSVICIPNNGQCFCLAWLKSNGTNAEKLAANILQWITFALSALCLMFYGYQTWKSTCGWETIYVATIEMIKFIIEYFHEFDEPAVIYSSNGNKTVWRYATWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGCIVWGATSAMCTGWTKILFFLISLSYGMYTYFHAAKVYIEAFHTVPKGICRELVRVMAWTFFVAWGMFPVLFLLGTEGFGHISPYGSAIGHSILDLIAKNMWGVLGNYLRVKIHEHILLYGDIRKKQKITIAGQEMEVETLVAEEE (Humanized C1V1 E122T/E162T nucleotide sequence)SEQ ID NO: 8atgtcgcgacgcccgtggctccttgctctcgcattggcggtggcgcttgcagcgggatcggcaggagcgtcaaccggaagcgatgcgaccgtccccgtggctacgcaagacggaccagattacgtgttccacagagcccacgagcggatgttgtttcagacatcatacacacttgaaaacaatggtagcgtcatttgcatccctaacaatgggcagtgtttttgcctggcctggttgaaatcgaacggtacgaacgccgagaagctggaggcgaacattctgcagtggatcacattcgcactcggcgctctgccttatgttctatggctaccagacttggaaatccacgtgtggttgggaaaccatctacgtagcaaccattgaaatgatcaagtttatcattgagtatttccatgagtttgacgaaccggccgtaatctactcatcgaatgggaataagacagtctggttgaggtatgcgacgtggctcctcacctgcccggtccttctgatccatctgagcaacctcacaggcctgaaggacgattatagcaaaaggactatgggcctgttggtttctgatgtgggatgcatcgtgtggggcgcaaccagcgccatgtgtacggggtggacgaagatcctgttcttcctcatctcattgagctatggtatgtatacctattttcatgctgctaaagtttatatcgaagcattccacacagttccaaaagggatttgtcgagaactggtccgagtgatggcctggacattctttgtggcttggggaatgtttccagtcctgtttctgctgggcacggaaggattcggtcatatcagcccttatggatctgccattgggcactccatcctcgacctgattgcaaagaacatgtggggtgtgctggggaattacctgcgcgtcaaaatccacgagcacatcctgttgtatggcgacatcagaaagaagcagaaaattacgatcgccggccaagagatggaggttgagacactggtggctgaagaggaggactaa(Alternative Humanized C1V1 amino acid sequence (C1V1_25)) SEQ ID NO: 9MSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERMLFQTSYTLENNGSVICIPNNGQCFCLAWLKSNGTNAEKLAANILQWITFALSALCLMFYGYQTWKSTCGWEEIYVATIEMIKFIIEYFHEFDEPATLWLSSGNGVVWMRYGEWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGCIVWGATSAMCTGWTKILFFLISLSYGMYTYFHAAKVYIEAFHTVPKGICRELVRVMAWTFFVAWGMFPVLFLLGTEGFGHISPYGSAIGHSILDLIAKNMWGVLGNYLRVKIHEHILLYGDIRKKQKITIAGQEMEVETLVAEEED(Alternative Humanized C1V1 nucleotide sequence (C1V1_25)) SEQ ID NO: 10AtgagcagacggccctggctgctggccctggctctcgctglggccaggccgccggcagcgccggagccagcaccggcagcgacgccaccgtgcccgttgccacacaggacggccccgactaCgtgttCcaccgggcccacgageggatgctgttccagaccagctacacccttgaaaacaacggcagcgtgatctgcatccccaacaacggccagtgettctgcctggcctggctgaagtccaacggcaccaacgccgagaagctggccgccaacatcctgcagtggatcaccttcgccctgtctgccctgtgcctgatgttctacggctaccagacctggaagtccacctgcggctgggaggaaatctacgtggccaccatcgagatgatcaagttcatcatcgagtacttccacgagttcgacgagcccgtaccctgtggctgtccagcggaaacggcgtggtgtggatgagatacggcgagtggctgctgacctgccctgtgctgctgatccacctgagcaacctgaccggactgaaggatgactacagcaagagaaccatgggactgctggtgtccgatgtgggatgcatcgtgtggggagccacctccgccatgtgcaccggatggaccaagatcctgttcttcctgatcagcctgagctacggaatgtacacctacttccacgccgccaaggtgtacattgaggcctttcacaccgtgcctaagggaatctgcagagaactggtcagagtgatggcctggaccttcttcgtggcctggggaatgttccctgtgctgttcctgctgggaaccgagggattcggacacatcagcccttacggaagcgccatcggacacagcatcctggatctgatcgccaagaacatgtggggagtgctgggaaactacctgagagtgaagatccacgagcacatcctgctgtacggcgacatcagaaagaagcagaagatcaccatcgccggacaggaaatggaagtcgagaccctgglggccgaggaagaggat ChR2 amino acid sequence SEQ ID NO: 11MDYGGALSAVGRELLFVTNPVVVNGSVINPEDQCYCAGWIESRGTNGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSIVILYLATGHRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTETEVETLV EDEAEAGAVPChR2(H134R) SEQ ID NO: 12MDYGGALSAVGRELLFVTNPVVVNGSVINPEDQCYCAGWIESRGTNGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLV EDEAEAGAVPSFO SEQ ID NO: 13MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQTASNVLQWLAAGESILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLYLATGHRVQWLRYAEWLLTSPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGEGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLVEDEAEAGAVP (SSFO) SEQ ID NO: 14MDYGGALSAVGRELLEVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQTASNVLQWLAAGESILLLMFYAYQTWKSTCGWEEIYVCALEMVKVILEFFFEEKNPSMLYLATGHRVQWLRYAEWLLTSPVILIHLSNLTGLSNDYSRRTMGLLVSAIGTIVWGATSAMATGYVKVIEFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLVEDEAEAGAVP

The invention claimed is:
 1. An isolated light-responsive chimericpolypeptide comprising an amino acid sequence having at least 95% aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:1.
 2. The chimeric polypeptide of claim 1, wherein the polypeptide isactivated by light of a wavelength between about 540 nm to about 560 nm.3. The chimeric polypeptide of claim 1, further comprising a C-terminaltrafficking signal.
 4. The chimeric polypeptide of claim 3, wherein thetrafficking signal comprises the amino acid sequenceKSRITSEGEYIPLDQIDINV (SEQ ID NO:15).
 5. The chimeric polypeptide ofclaim 1, wherein the light-responsive chimeric polypeptide comprises anamino acid sequence having at least 98% amino acid sequence identity tothe amino acid sequence set forth in SEQ ID NO:1.
 6. The chimericpolypeptide of claim 1, wherein the polypeptide comprises a Glu to Thramino acid substitution at position 122 relative to the amino acidsequence set forth in SEQ ID NO:1.
 7. The chimeric polypeptide of claim1, wherein the polypeptide comprises a Glu to Thr amino acidsubstitution at position 162 relative to the amino acid sequence setforth in SEQ ID NO:1.
 8. The chimeric polypeptide of claim 1, whereinthe polypeptide comprises a Glu to Thr amino acid substitution atposition 122 and a Glu to Thr amino acid substitution at position 162relative to the amino acid sequence set forth in SEQ ID NO:1.
 9. Thechimeric polypeptide of claim 1, wherein the light-responsive chimericpolypeptide comprises an amino acid sequence having at least 99% aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:1.
 10. An isolated polynucleotide comprising a nucleotide sequenceencoding a light-responsive chimeric polypeptide comprising an aminoacid sequence having at least 95% amino acid sequence identity to theamino acid sequence set forth in SEQ ID NO:1.
 11. The isolatedpolynucleotide of claim 10, wherein the nucleotide sequence is operablylinked to a promoter.
 12. The polynucleotide of claim 10, wherein thepolynucleotide is an expression vector.
 13. The polynucleotide of claim12, wherein the expression vector is a viral vector.
 14. The isolatedpolynucleotide of claim 10, wherein the light-responsive chimericpolypeptide comprises an amino acid sequence having at least 98% aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:1.
 15. The isolated polynucleotide of claim 10, wherein thelight-responsive chimeric polypeptide further comprises a C-terminaltrafficking signal.
 16. The isolated polynucleotide of claim 15, whereinthe trafficking signal comprises the amino acid sequenceKSRITSEGEYIPLDQIDINV (SEQ ID NO:15).
 17. The isolated polynucleotide ofclaim 10, wherein the light-responsive chimeric polypeptide comprises aGlu to Thr amino acid substitution at position 122 relative to the aminoacid sequence set forth in SEQ ID NO:1.
 18. The isolated polynucleotideof claim 10, wherein the light-responsive chimeric polypeptide comprisesa Glu to Thr amino acid substitution at position 162 relative to theamino acid sequence set forth in SEQ ID NO:1.
 19. The isolatedpolynucleotide of claim 10, wherein the light-responsive chimericpolypeptide comprises a Glu to Thr amino acid substitution at position122 and a Glu to Thr amino acid substitution at position 162 relative tothe amino acid sequence set forth in SEQ ID NO:1.